Runx2-positive Cells Research Articles

Lysyl oxidase-dependent extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease

Extracellular matrix (ECM) is an active player in cardiovascular calcification (CVC), a major public health issue with an unmet need for effective therapies. Lysyl oxidase (LOX) conditions ECM biomechanical properties; thus, we hypothesized that LOX might impact on mineral deposition in calcific aortic valve disease (CAVD) and atherosclerosis. LOX was upregulated in calcified valves from two cohorts of CAVD patients. Strong LOX immunostaining was detected surrounding calcified foci in calcified human valves and atherosclerotic lesions colocalizing with RUNX2 on valvular interstitial cells (VICs) or vascular smooth muscle cells (VSMCs). Both LOX secretion and organized collagen deposition were enhanced in calcifying VICs exposed to osteogenic media. β-aminopropionitrile (BAPN), an inhibitor of LOX, attenuated collagen deposition and calcification. VICs seeded onto decellularized matrices from BAPN-treated VICs calcified less than cells cultured onto control scaffolds; instead, VICs exposed to conditioned media from cells over-expressing LOX or cultured onto LOX-crosslinked matrices calcified more. Atherosclerosis was induced in WT and transgenic mice that overexpress LOX in VSMC (TgLOXVSMC) by AAV-PCSK9D374Y injection and high-fat feeding. In atherosclerosis-challenged TgLOXVSMC mice both atherosclerosis burden and calcification assessed by near-infrared fluorescence (NIRF) imaging were higher than in WT mice. These animals also exhibited larger calcified areas in atherosclerotic lesions from aortic arches and brachiocephalic arteries. Moreover, LOX transgenesis exacerbated plaque inflammation, and increased VSMC cellularity, the rate of RUNX2-positive cells and both connective tissue content and collagen cross-linking. Our findings highlight the relevance of LOX in CVC and postulate this enzyme as a potential therapeutic target for CVC.

Effects of Achyranthes bidentata alcohol on proliferation capacity of osteoblasts and miRNA in Runx2.

Achyranthes bidentata is a herbal plant commonly used in the treatment of osteoporosis and bone nonunion with traditional Chinese medicine. Achyranthes bidentata alcohol is a major component extracted from Achyranthes bidentata, which has been proved to be able to exert a variety of pharmacological effects, such as anti-inflammation, antipyresis, anti-rheumatism, diuresis and anti-osteoporosis. Thirty male Sprague-Dawley rats aged 4 weeks were used in the experiment. All primary rat osteoblasts were cultured and amplified for further experiments. The osteoblasts were divided into six groups (5 rats in each group): the culture medium control group, the 25 µg/ml achyranthol group, the 50 µg/ml achyranthol group, the 100 µg/ml achyranthol group, 200 µg/ml achyranthol group, and the 25 µM PD98059+200 µg/ml achyranthol group. In this study, the effect of Achyranthes bidentata alcohol on the proliferation of osteoblasts was detected via methyl thiazolyl tetrazolium (MTT) assay. The effect of Achyranthes bidentata alcohol on the alkaline phosphatase (ALP) activity in osteoblasts was analyzed via ALP assay. The effect of Achyranthes bidentata alcohol on the expression of osteoblast marker gene, Runt-related transcription factor 2 (Runx2), was detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Moreover, the phosphorylation or activation of extracellular signal-regulated kinase (ERK) in osteoblasts induced by Achyranthes bidentata alcohol was analyzed using western blotting. Achyranthes bidentata alcohol increased cell proliferation in a dose-dependent manner, increased the micro ribonucleic acid (miRNA) level in Runx2, enhanced the ALP activity in osteoblasts, and stimulated the activation of ERK (P<0.05). The expression of Runx2 with the inhibitor PD98059 was decreased significantly compared with that in the Achyranthes bidentata alcohol group (P<0.01). Immunohistochemical results manifested that the percentage of Runx2 positive cells in treated tissues was obviously higher than that in untreated tissues (P<0.01). Therefore, Achyranthes bidentata alcohol promotes the proliferation capacity of osteoblasts in a dose-dependent manner, enhances the expression of miRNA in Runx2, and stimulates the osteogenic differentiation of osteoblasts through activating the ERK signal transduction pathway.

Vital Bone Formation After Grafting of Autogenous Bone and Biphasic Calcium Phosphate Bioceramic in Extraction Sockets of Rats: Histological, Histometric, and Immunohistochemical Evaluation.

The study aimed to investigate through histology, histometry, and immunohistochemistry the vital bone formation after grafting of biphasic calcium phosphate bioceramic (BC) in combination with calvarial autogenous bone into the dental sockets of rats. Forty-five male rats were submitted to upper right incisor extraction and divided according to the grafted material in: control, bioceramic (BC), and bioceramic + autogenous bone (BC + AB). The animals were killed 7, 21, and 42 days after surgery for histological, histometric, and immunohistochemistry analysis. Histomorphometric results demonstrated, for BC + AB group, formation of trabecular bone between the particles of BCs and autogenous bone, connecting them, as well as higher percentage of vital bone in comparison with BC. Immunohistochemical reactions showed intense labeling for Runx2-positive cells in the group BC + AB. Autogenous bone was able to stimulate bone turnover enabling a larger amount of vital bone synthesis and can be recommended as a viable grafting material in combination with synthetic biphasic BC.

Enhancement of Bone Augmentation in Osteoporotic Conditions by the Intermittent Parathyroid Hormone: An Animal Study in the Calvarium of Ovariectomized Rat.

Intermittent parathyroid hormone (PTH) is the commonly used therapeutic approach for patients with severe osteoporosis. The goal of this study was to elucidate the effect of the intermittent PTH treatment on guided bone augmentation (GBA) in the calvarium of ovariectomized (OVX) rats. Surgical ovariectomy on 14 rats and sham surgery on 7 rats were conducted on all rats as the first surgery. GBA surgery was conducted 8 weeks following the first surgery in the rat calvarium by placing 5-mm-diameter cylindrical plastic caps. Following surgery, rats were treated with 40 μg/kg PTH (OVX-PTH) or saline (Sham-Saline, OVX-Saline) via intraperitoneal injection three times per week during the all-observational period. Longitudinal microcomputed tomography (micro-CT) imaging was performed every 2 weeks following the GBA surgery without euthanasia, and the amount of newly generated bone volume (BV) was calculated. All rats were euthanized 12 weeks after GBA surgery, and histology was obtained. Sections stained with hematoxylin and eosin were used for the quantitative analysis of newly generated tissue, and immunohistology was used to visualize Runx2-positive cells and TRAP-positive cells. Throughout the monitoring period, the BVs of OVX rats without PTH treatment (OVX-Saline) were significantly lower than that of the other two groups at weeks 8 and 12 in micro-CT analysis. During all experimental periods, the BV was highest in the OVX rats that were treated with PTH (OVX-PTH). Histologic analysis confirmed the result of micro-CT, and determined that the OVX-PTH presented a greater number of Runx2-positive cells. The number of TRAP-positive multinucleated osteoclasts was highest in OVX-PTH rats; there were no significant differences between the other two groups. The results of this study suggest that treatment with intermittent PTH was associated with increased newly regenerated bone volume in ovariectomized rat calvarial bone augmentation, which may have important clinical implications.

Setd7 and its contribution to Boron-induced bone regeneration in Boron-mesoporous bioactive glass scaffolds

Boron (B), a trace element found in the human body, plays an important role for health of bone by promoting the proliferation and differentiation of osteoblasts. Our research group previously fabricated B-mesoporous bioactive glass (MBG) scaffolds, which successfully promoted osteogenic differentiation of osteoblasts when compared to pure MBG scaffolds without boron. However, the mechanisms of the positive effect of B-MBG scaffolds on osteogenesis remain unknown. Therefore, we performed in-vivo experiments in OVX rat models with pure MBG scaffolds and compared them to B-MBG scaffold. As a result, we found that B-MBG scaffold induced more new bone regeneration compared to pure MBG scaffold and examined genes related to bone regeneration induced by B-MBG scaffold through RNA-seq to obtain target genes and epigenetic mechanisms. The results demonstrated an increased expression and affiliation of Setd7 in the B-MBG group when compared to the MBG group. Immunofluorescent staining from our in vivo samples further demonstrated a higher localization of Setd7 and H3K4me3 in Runx2-positive cells in defects treated with B-MBG scaffolds. KEGG results suggested that specifically Wnt/β-catenin signaling pathway was highly activated in new bone area associated with B-MBG scaffolds. Thereafter, in vitro studies with human bone marrow stem cells (hBMSCs) stimulated by extracted liquid of B-MBG scaffolds was associated with significantly elevated levels of Setd7, as well as H3K4me3 when compared to MBG scaffolds alone. To verify the role of Setd7 in new bone formation in the presence of Boron, Setd7 was knocked down in hBMSCs with stimulation of the extracted liquids of B-MBG or MBG scaffolds. The result showed that osteoblast differentiation of hBMSCs was inhibited when Setd7 was knocked down, which could not be rescued by the extracted liquids of B-MBG scaffolds confirming its role in osteoblast differentiation and bone regeneration. As a histone methylase, Setd7 may be expected to be a potential epigenetic target for new treatment schemes of osteoporosis. Statement of SignificanceBoron-containing MBG scaffold has already been proved to promote bone regeneration in femoral defects of OVX rats by our research group, however, the epigenetic mechanism of Boron’s positive effects on bone generation remains ill-informed. In our present study, we found an increased expression and affiliation of Setd7 and H3K4me3 in Runx2-positive osteoblasts in vivo. And in vitro, the higher expression of Setd7 enhanced osteogenic differentiation of human BMSCs stimulated by extracted liquids of B-MBG scaffold compared to MBG scaffold, which was associated with the activation of Wnt/β-catenin signaling pathway. Above all, it suggests that Setd7 plays an positive role in osteogenic differentiation and it may become a potential epigenetic target for new schemes for osteoporosis.

Osteogenic cellular activity around onlaid octacalcium phosphate‐gelatin composite onto rat calvaria

The activity and the distribution of osteogenic cells around octacalcium phosphate (OCP) granules combined with gelatin matrix (OCP/Gel) onlaid on rat calvaria were analyzed histomorphometrically and immunohistochemically during vertical bone augmentation under mechanical or nonmechanical environment until 8 weeks. OCP/Gel disk was placed in subperiosteal pocket on the calvaria with or without polytetrafluoroethylene (PTFE) support. The latter is a nonmechanical stress model to alleviate the mechanical stress from the subcutaneous tissues. Onlay grafts of gelatin (Gel) sponge disk and OCP granules were also carried out for the comparison purpose. When bone augmentation was evaluated in first area from bone surface (area until 150 μm high from bone surface) and second area above the newly formed bone (area until 150 µm high from the first area), bone formation was enhanced most in first area followed by second area of OCP/Gel with PTFE. The appearance of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells was suppressed more in the newly formed tissue with PTFE than those without PTFE with an emphasis of the presence of gelatin. Although Runx2 positive-cells were accumulated more in both OCP/Gel with and without PTFE, osteocalcin-positive cells were abundant in OCP/Gel with PTFE than that without PTFE, suggesting that nonmechanical stress condition is more suitable for osteoblast differentiation. The appearance of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-positive cells was restrained in OCP/Gel and Gel with PTFE while osteoprotegerin-positive cells were most accumulated in OCP/Gel without PTFE. The results suggest that OCP/Gel composite under the mechanical stress-alleviated condition can enhance bone augmentation through balanced osteoblastic and osteoclastic cellular activities. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1322-1333, 2018.

Effects of combined therapy of alendronate and low-intensity pulsed ultrasound on metaphyseal bone repair after osteotomy in the proximal tibia of glucocorticoid-induced osteopenia rats

ObjectivesGlucocorticoid (GC) treatment inhibits activation of runt-related transcription factor 2 (Runx2), which is essential for osteoblast differentiation from stem cells. As a result, GC treatment results in bone loss, GC-induced osteoporosis (GIO), elevated fracture risk, and delayed bone healing. Bisphosphonates such as alendronate (ALN) are recommended for treating or preventing GIO, and low-intensity pulsed ultrasound (LIPUS) facilitates fracture healing and maturation of regenerated bone. Combined therapy with ALN and LIPUS may stimulate cancellous bone healing in GIO rats. Here, we examined the effect of ALN and LIPUS on cancellous bone osteotomy repair in the proximal tibia of GIO rats. MethodsPrednisolone (10 mg/kg body weight/day) was administered for 4 weeks to induce GIO in 6-month-old female Sprague-Dawley rats. Tibial osteotomy was then performed and daily subcutaneous injection of ALN (1-μg/kg body weight) was subsequently administered alone or in combination with LIPUS (20 min/day) for 2 or 4 weeks. ResultsALN significantly increased bone mineral density (BMD) at 2 and 4 weeks, and ALN + LIPUS significantly increased BMD at 4 weeks. Bone union rates were significantly increased after 2 and 4 weeks ALN and ALN + LIPUS treatment. Lastly, ALN and ALN + LIPUS significantly increased the proportion of Runx2 positive cells at 4 weeks. ConclusionsALN monotherapy and combined ALN and LUPUS treatment augmented BMD and stimulated cancellous bone repair with increased Runx2 expression at the osteotomy site in GIO rats. However, the combined treatment had no additional effect on cancellous bone healing compared to ALN monotherapy.

The sp7 gene is required for maturation of osteoblast-lineage cells in medaka (Oryzias latipes) vertebral column development

Sp7 is a zinc finger transcription factor that is essential for osteoblast differentiation in mammals. To verify the characteristic features of osteoblast-lineage cells in teleosts, we established medaka sp7 mutants using a transcription activator-like effector nuclease (TALEN) genome editing system. These mutants showed severe defects in the formation of skeletal structures. In particular, the neural and the hemal arches were not formed, although the chordal centra were formed. Analysis of the transgenic medaka revealed that sp7 mutant had normal distribution of type X collagen a1 a (col10a1a)-positive osteoblast-like cells around the centrum and at the proximal region of the vertebral arch. The sp7 mutant phenotype could be rescued by exogenous sp7 expression in col10a1a-positive cells, as well as in sp7-positive osteoblast cells. Furthermore, runx2-positive osteoblast progenitors were observed on the vertebral arches, but not on the centrum, during vertebral column development. In addition, these osteoblast progenitors differentiated into the col10a1a-positive cells. In sp7 mutant, the runx2-positive cells were normally distributed at the region of unformed vertebral arch but failed to differentiate into col10a1a-positive cells. These results indicate that osteoblast-lineage cells undergo two distinct differentiation processes during development of the vertebral arch and the centrum. Nevertheless, our results verified that sp7 gene expression in osteoblast-lineage cells is required for differentiation into mature osteoblasts to form the vertebral column and other skeletal structures.

Setd2 is associated with strontium-induced bone regeneration.

Our research group recently demonstrated that the combination of MBG scaffolds with Sr, efficiently promoted bone regeneration in rat femoral defects even in severely compromised osteoporotic animals, however, the epigenetic mechanism by which Sr ions function to promote bone generation remains unclear. This study showed an increased expression and affiliation of Setd2 and H3K36me3. In vitro, the increased expression of Setd2 promoted osteoblastic differentiation of MG63 stimulated by SrCl2 in MAPK-dependent way, which activated ERK in turn leading to a positive feedback. Based on these findings, it was shown that Setd2 has an active role in osteoblast differentiation and may also turn to be an epigenetic target for new treatment options of osteoporosis and the development of novel bone regeneration scaffold.

Abstract 647: Induction of Cardiovascular Calcification in Non-transgenic Mice via a Single Injection of Pcsk9 Adeno-associated Viral Vector

Background: Studying atherosclerotic calcification in vivo requires mouse models with genetic deletion of low-density lipoprotein receptor (Ldlr) or apolipoprotein E. A previous study showed a rapid induction of atherosclerosis by proprotein convertase subtilisin/kexin type 9 (PCSK9) in mice. Here, we hypothesize that this method is a useful in vivo tool to study cardiovascular calcification in non-genetically modified C57BL/6 mice. Results: 10 week old C57BL/6 mice received a single tail vein injection of recombinant adeno-associated viral vector (AAV) encoding PCSK9 (rAAV8/D377Y-mPCSK9). Ldlr -/- and saline injected C57BL/6 mice served as controls. Mice consumed a high-fat, high-cholesterol (HF/HC) diet for 15-20 weeks. PCSK9 and total cholesterol serum levels were significantly increased within one week after injection and maintained for 20 weeks (cholesterol: 82 mg/dL to 820 mg/dL, p&lt;0.01; PCSK9: 0.14 μg/ml to 20 μg/ml, p&lt;0.01). Total cholesterol levels remained 20-30% lower than those of of Ldlr -/- mice. Atherosclerotic lesion size was similar between PSCK9 and Ldlr -/- mice. Saline injected mice did not show any lesions. Plaque collagen content was 31.9%±6.6 in PCSK9 mice and 62.9%±16.6 in Ldlr -/- mice at 15 weeks of HF/HC diet (p=0.01). However, by 20 weeks, the PCSK9 mice had 57.9%±18.6 plaque collagen, suggesting a different stage of plaque progression. Fluorescence reflectance imaging of a near infrared calcium tracer in intact arteries detected 0.4%±0.4 aortic calcification in PCSK9 mice and 9.7%±1.6 in Ldlr -/- mice at 15 weeks of HF/HC diet (p=0.01); by 20 weeks, the PCSK9 mice had 5.3%±1.0 aortic calcification. Tissue non-specific alkaline phosphatase activity positive lesion area was 7.9%±4.0 and 8.3%±2.6 in PCSK9 mice and 10.8%±2.5 and 12.7%±1.7in Ldlr -/- mice at 15 and 20 weeks, respectively. Immunofluorescence analysis demonstrated accumulation of CD68 and RUNX2-positive cells in the plaques of PCSK9 mice similar to Ldlr -/- . Conclusion: While injection of recombinant AAV encoding PCSK9 into C57BL/6 mice induces atherosclerotic calcification with slower sclerotic plaque remodeling compared to Ldlr -/- mice, it may serve as a useful tool to study cardiovascular calcification in mice independent of their genetic background.

Abstract P5-05-02: Regulation of glucose metabolism by the RUNX2 transcription factor has a negative impact on mitochondrial function in breast cancer cells

Abstract The RUNX2 transcription factor regulates breast cancer (BC) metastasis to bone and is itself a target of IGF-1 signaling pathways. We have shown that glucose can activate RUNX2 phopshorylation through IGF-1 receptor signaling and that RUNX2 was associated with inhibition of pyruvate dehydrogenase (PDHA1) activity and repression of mitochondrial respiration in BC cells. However, the mechanisms by which RUNX2 alters mitochondrial function and supports an oncogenic phenotype are not completely known. RUNX2 expression in a luminal BC cell line increased expression of glycolytic genes, and sensitivity to glucose starvation. However, RUNX2 knockdown in a triple-negative BC cell line inhibited expression of glycolytic genes. RUNX2 also repressed mitochondrial oxygen consumption rates (OCR), a measure of oxidative phosphorylation while overexpression of SIRT6, a NAD-dependent histone deacetylase, increased respiration in RUNX2-positive cells. RUNX2 repressed SIRT6 expression directly at the transcriptional and post-translational levels. High SIRT6 expression was observed in normal mammary tissue and cells that did not express RUNX2 but endogenous SIRT6 expression was low in malignant BC tissues or cell lines, which expressed high levels of RUNX2. These results suggest that SIRT6, a known tumor suppressor, was a critical mediator of these RUNX2-regulated metabolic changes. These results support a hypothesis whereby RUNX2 upregulation during BC progression leads to inactivation of the SIRT6 tumor suppressor to promote tumorigenesis. It will be important to investigate further changes in mitochondrial function to understand the precise mechanisms by which RUNX2 regulates BC metabolism. Citation Format: Kim MS, Choe M, Cho H, Polster BM, Girnun GD, Passaniti A. Regulation of glucose metabolism by the RUNX2 transcription factor has a negative impact on mitochondrial function in breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-05-02.

Recombinant osteoprotegerin effects during orthodontic movement in a rat model.

Anchorage is one of the most challenging sides in orthodontics. The use of biological modulators that inhibit osteoclasts could be a solution to address these problems and provide new adjunctive approaches. The aim of this study was to assess the effectiveness of recombinant osteoprotegerin fusion protein (OPG-Fc) in orthodontic anchorage. Two groups of male Sprague-Dawley rats were utilized. The animals in the experimental group received twice-weekly injections with high dose of OPG-Fc (5.0mg/kg) in mesial and distal mucosa of the first molars, and those in the control group received no drugs. Right first maxillary molars were mesialized using a calibrated nickel-titanium spring connected to an anterior mini-screw. Tooth movement was measured by two blinded observers using scanned and magnified stone casts. Receptor activator of nuclear factor κB (RANK), run-related transcription factor 2 (Runx2), type I collagen, vimentin, matrix metalloproteinases 2 and 9, S100 protein and the putative mechanoproteins acid-sensing ion channel (ASIC2) and transient receptor potential vainilloid 4 (TRPV4) were evaluated using immunohistochemistry. OPG-Fc group showed an important decreased in mesial molar movement with only 52%, 31%, and 22% of the total mesial molar movement compared with control group at Days 7, 14, and 21, respectively (P < 0.001). RANK ligand and Runx2 positive cells were severely reduced after OPG-Fc treatment. Periodontal ligament architecture, cell arrangement, and immunohistochemical patter for vimentin, type I collagen and the mechanoproteins TRPV4 and ASIC2 were altered by tooth movement and all these parameters altered by the applied treatment. OPG-Fc effectively inhibits osteoclastogenesis resulting in improved bone quantity and orthodontic anchorage. Based on present results, OPG-Fc could have clinical utility in preventing undesired tooth movements.

The RUNX2 Transcription Factor Negatively Regulates SIRT6 Expression to Alter Glucose Metabolism in Breast Cancer Cells.

Activation of genes promoting aerobic glycolysis and suppression of mitochondrial oxidative phosphorylation is one of the hallmarks of cancer. The RUNX2 transcription factor mediates breast cancer (BC) metastasis to bone and is regulated by glucose availability. But, the mechanisms by which it regulates glucose metabolism and promotes an oncogenic phenotype are not known. RUNX2 expression in luminal BC cells correlated with lower estrogen receptor-α (ERα) levels, anchorage-independent growth, expression of glycolytic genes, increased glucose uptake, and sensitivity to glucose starvation, but not to inhibitors of oxidative phosphorylation. Conversely, RUNX2 knockdown in triple-negative BC cells inhibited mammosphere formation and glucose dependence. RUNX2 knockdown resulted in lower LDHA, HK2, and GLUT1 glycolytic gene expression, but upregulation of pyruvate dehydrogenase-A1 (PDHA1) mRNA and enzymatic activity, which was consistent with lower glycolytic potential. The NAD-dependent histone deacetylase, SIRT6, a known tumor suppressor, was a critical regulator of these RUNX2-mediated metabolic changes. RUNX2 expression resulted in elevated pAkt, HK2, and PDHK1 glycolytic protein levels that were reduced by ectopic expression of SIRT6. RUNX2 also repressed mitochondrial oxygen consumption rates (OCR), a measure of oxidative phosphorylation (respiration). Overexpression of SIRT6 increased respiration in RUNX2-positive cells, but knockdown of SIRT6 in cells expressing low RUNX2 decreased respiration. RUNX2 repressed SIRT6 expression at both the transcriptional and post-translational levels and endogenous SIRT6 expression was lower in malignant BC tissues or cell lines that expressed high levels of RUNX2. These results support a hypothesis whereby RUNX2-mediated repression of the SIRT6 tumor suppressor regulates metabolic pathways that promote BC progression.

Enhancement of Runx2 expression is potentially linked to β-catenin accumulation in canine intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) greatly affects the quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP because the cells disappear with age and are replaced by fibrochondrocyte-like cells. Because IVDD develops as early as within the first year of life, we used canines as a model to investigate the in vitro mechanisms underlying IVDD. The mechanism underlying age-related IVDD, however, is poorly understood. Several research groups have suggested that Wnt/β-catenin signaling plays an important role in IVDD. However, the role of Wnt/β-catenin signals in IVD cells is not yet well understood. Here, we demonstrate that Wnt/β-catenin signaling could enhance Runx2 expression in IVDD and lead to IVD calcification. Nucleus pulposus (NP) tissue was obtained from Beagle dogs after evaluation of the degeneration based on magnetic resonance imaging (MRI). Histological analysis showed that lack of Safranin-O staining, calcified area, and matrix metalloproteinase (MMP) 13-positive cells increased with progression of the degeneration. Furthermore, the levels of β-catenin- and Runx2-positive cells also increased. Real-time reverse-transcription polymerase chain reaction analysis showed that the MRI signal intensity and mRNA expression levels of β-catenin and Runx2 are correlated in NP tissues. Moreover, supplementation of LiCl induced β-catenin accumulation and Runx2 expression. In contrast, FH535 inhibited LiCl-induced upregulation. These results suggest that Runx2 transcript and protein expression, potentially in combination with β-catenin accumulation, are enhanced in degenerated and calcified intervertebral discs of CDBs.

Effectiveness of the diode laser in the treatment of ligature-induced periodontitis in rats: a histopathological, histometric, and immunohistochemical study.

The aim of this study was to evaluate the effects of the use of a high-power gallium-aluminum-arsenide diode laser (GaAlAs; 808 nm, 1 W, 20 s, 20 Hz, 10 J) alone or as adjunctive therapy to scaling and root planing in the treatment of induced periodontitis in rats. Periodontitis was induced by placing a ligature around the mandibular first molar of 60 rats. After 7 days, the ligature was removed and the animals were divided into four groups as follows: C (control), no periodontal treatment; SRP, scaling and root planing (SRP); DL, diode laser (DL) irradiation treatment; and SRP/DL, both SRP and DL irradiation treatment. Five animals from each group were euthanized at 7, 15, and 30 days posttreatment. The effectiveness of the treatments was evaluated in the furcation area using histopathological analysis, histometric analysis of alveolar bone loss (ABL), and immunohistochemical detection of tartrate-resistant acid phosphatase (TRAP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). DL, alone or in combination with adjunctive therapy to SRP in the treatment of experimental periodontitis, resulted in a decreased local inflammatory response. At 7-days posttreatment, the DL and SRP/DL groups had fewer TRAP-positive cells and more RUNX2-positive cells. There was greater OCN immunolabeling in the DL group than in the C and SRP groups at 15 days. There was less ABL in the DL and SRP/DL groups at 15 and 30 days. In conclusion, DL was effective in the treatment of ligature-induced periodontitis in rats, both when used alone and when used as adjunctive therapy to SRP.

Bone marrow aspirate combined with low-level laser therapy: A new therapeutic approach to enhance bone healing

This study evaluated the influence of bone marrow aspirate (BMA), low-level laser therapy (LLLT) and their combination on bone healing in surgically created critical-size defects (CSDs) in rat calvaria. 40 rats were divided into four groups: C (control), BMA, LLLT and BMA/LLLT. A 5mmdiameter CSD was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group BMA, the defect was filled with BMA. In groups LLLT and BMA/LLLT, the defect received laser irradiation (InGaAlP laser), was filled with blood clot or BMA respectively, and irradiated again. Animals were euthanized 30days postoperatively. Histomorphometric and immunohistochemical analyses were performed. Newly formed bone area (NFBA) was calculated as percentage of the total area of the original defect. Proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) immunohistochemical staining were performed. PCNA-positive, Runx2-positive and OCN-positive cells were quantified. Data were statistically analyzed. Group BMA/LLLT had significantly greater NFBA than groups C, BMA or LLLT. Group BMA presented significantly greater NFBA than control, while group LLLT did not. Group BMA/LLLT presented a significantly higher number of PCNA-positive and OCN-positive cells than any of the other groups. Groups BMA/LLLT and BMA showed a significantly lower number of Runx2-positive cells than groups C or LLLT. The combination of BMA/LLLT yielded significantly greater bone formation in surgically created CSD in rat calvaria when compared to control, or either treatment alone.

Evaluation of Cameroonian plants towards experimental bone regeneration

Ethnopharmacological relevanceElephantopus mollis, Spilanthes africana, Urena lobata, Momordica multiflora, Asystasia gangetica and Brillantaisia ovariensis are used in Cameroonian traditional medicine for the treatment of bone diseases and fracture repair. The aim of this study was to evaluate the effect of ethanolic extracts of six Cameroonian medicinal plants on bone regeneration following bone and marrow injury. Materials and methodsEthanol extract of Cameroonian medicinal plants were administered (each extract at 250, 500 and 750mg/kg doses) orally to adult female Sprague–Dawley rats having a drill hole injury (0.8mm) in the femur diaphysis. Vehicle (gum-acacia in distilled water) was given to the control group. After 12 days of treatment, animals were euthanized and femur bones collected. Confocal microscopy of fractured bone was performed to evaluate bone regeneration (calcein labeling). Only active plant extracts were used for further experiments. Thus, callus was analyzed by microcomputed tomography. Osteogenic effects of the extracts were evaluated by assessing mineralized nodules formation of bone marrow stromal cells and osteoblast recruitment at drill hole site by immunohistochemistry. ResultsEthanolic extract of the leaves and twigs of Elephantopus mollis (EM) and whole plant of Spilanthes africana (SA) dose-dependently stimulated bone regeneration at the drill hole site. EM at 250 and 750mg/kg doses and SA at 750mg/kg dose significantly increased mineral deposition compared to controls. Both extracts at 500 and 750mg/kg doses improved microarchitecture of the regenerating bone evident from increased bone volume fraction, trabecular thickness, trabecular number, and decreased trabecular separation and structure model index. EM and SA extracts increased the formation of mineralized nodules from the bone marrow stromal cells. In addition, EM and SA extracts increased osteoblast recruitment at the drill hole site evident from increased Runx-2 positive cells following their treatments compared to control. ConclusionEthanolic extracts of EM and SA accelerate fracture repair in rats via stimulatory effects on osteoblast differentiation and mineralization, thereby justifying their traditional use.

Abstract P4-03-05: The Warburg Effect and RUNX2-Regulated Metabolic Switching in Breast Cancer: A New Therapeutic Target?

Abstract Breast cancer (BC) is the leading cause of cancer deaths among women in North America and Western Europe. Numerous studies have shown that breast tumor progression is associated with a switch from mitochondrial oxidative phosphorylation (OxPhos) to increased utilization of glucose and aerobic glycolysis (Warburg effect). Uncovering underlying mechanisms regulating this progression may lead to innovative approaches for treatment. The RUNX2 DNA-binding transcription factor is expressed in highly aggressive BC and regulates bone metastasis in animal models. We observed increased expression of RUNX2 in primary human ductal carcinoma in situ (DCIS), which correlated with grade and proliferative index (Ki67 staining), decreased estrogen receptor/progesterone receptor expression (ER/PR status), increased glucose uptake values by PET scanning, and increased expression of the glucose transporter, GLUT1. Further, more than 80% (9 out of 11) of triple-negative BC cell lines examined expressed RUNX2. Therefore, to test the hypothesis that RUNX2 may alter the metabolic status of breast cancers and increase their dependence on glycolysis during tumor progression, we have generated a Tet-OFF model system using MCF7 cells with inducible RUNX2 expression. RUNX2-positive MCF7 cells exhibited increased cell death under conditions of glucose depletion or when treated with 2-deoxy-glucose, a competitive inhibitor of hexokinase II. Conversely, RUNX2-positive cells were more resistant to OxPhos inhibitors (rotenone, oligomycin) than RUNX2-negative cells. We also observed higher expression levels of GLUT1 and pAKT in RUNX2-positive MCF7 cells. Hypoxia-inducible factor-α (HIF1α) is a transcription factor that regulates cancer metabolism through its activation of glycolytic genes, while SIRT6, an NAD-dependent deacetylase, is a negative regulator of HIF1α activity. This occurs, in part, via inhibition of HIF1α transcriptional activity. RUNX2-positive or negative cells expressed HIF1α in response to glucose but RUNX2-positive cells exhibited lower SIRT6 expression than RUNX2-negative cells, suggesting that with the loss of SIRT6, HIF1α might be more active in promoting glycolysis in the presence of RUNX2. These results suggest that RUNX2 may have clinical diagnostic value as an indicator of BC metabolic stress and predictor of poor prognosis. Current strategies are utilizing computer-assisted drug design to develop RUNX-specific inhibitors to target BC glycolysis and tumor growth. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-03-05.

Differentiation-dependent association of phosphorylated extracellular signal-regulated kinase with the chromatin of osteoblast-related genes

The ERK/MAP kinase pathway is an important regulator of gene expression and differentiation in postmitotic cells. To understand how this pathway controls gene expression in bone, we examined the subnuclear localization of P-ERK in differentiating osteoblasts. Induction of differentiation was accompanied by increased ERK phosphorylation and expression of osteoblast-related genes, including osteocalcin (Bglap2) and bone sialoprotein (Ibsp). Confocal immunofluorescence microscopy revealed that P-ERK colocalized with the RUNX2 transcription factor in the nuclei of differentiating cells. Interestingly, a portion of this nuclear P-ERK was directly bound to the proximal promoter regions of Bglap2 and Ibsp. Furthermore, the level of P-ERK binding to chromatin increased with differentiation, whereas RUNX2 binding remained relatively constant. The P-ERK-chromatin interaction was seen only in RUNX2-positive cells, required intact RUNX2-selective enhancer sequences, and was blocked with MAPK inhibition. These studies show for the first time that RUNX2 specifically targets P-ERK to the chromatin of osteoblast-related genes, where it may phosphorylate multiple substrates, including RUNX2, resulting in altered chromatin structure and gene expression. © 2010 American Society for Bone and Mineral Research

Proliferation and differentiation of periodontal ligament cells following short-term tooth movement in the rat using different regimens of loading

Previous studies have indicated that periodontal ligament (PDL) cells demonstrate osteogenic potential and osteoblastic differentiation via the extracellular signal-regulated kinase (ERK) pathway under mechanical stress in vitro and in vivo. This study aimed to further analyse this regulatory process experimentally in the rat. The right upper first molars of 25 twelve-week-old male Wistar anaesthetized rats were loaded with forces in order to be moved mesially. Constant forces for 4 hours of 0.25 and 0.5 N were applied in five animals each. Furthermore, constant forces for 2 hours of 0.1 N were applied in 10 animals and afterwards, the first and second molars were permanently separated with composite. In these animals, the antagonists were sliced and five rats were killed after 1 day and five after 2 days. As a last experiment, intermittent forces of 0.1 N and 0.25 Hz were applied in five different animals for 4 hours. The untreated contralateral sides served as the control. Paraffin-embedded sections were analysed by immunohistochemistry for proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2/Cbfa1), and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2). Statistical analysis to determine differences between the groups was carried out using a Student's t-test. In selected areas under tension, the proportion of pERK1/2-positive cells was increased compared with those in control teeth under all types of loading, whereas these proportions in selected areas under pressure were increased only after the application of intermittent forces. In representative areas, both under tension and pressure, the proportion of Runx2-positive cells decreased after the application of constant forces. After the application of constant forces for 4 hours in representative areas, both under tension and pressure, the proportion of PCNA-positive cells was lower than those in control teeth. The involvement of pERK1/2, Runx2/cbfa-1, and PCNA in the reaction of PDL cells to different load regimens was verified.