text:
Hypertrophy is induced during the in vitrochondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer
DOI: 10.1186/ar2822
© Steinert et al.; licensee BioMed Central Ltd. 2009
Received: 1 May 2009
Accepted: 2 October 2009
Published: 2 October 2009
The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.
Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 × 102 viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.
Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size.
BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.
Mesenchymal progenitor cells, also referred to as mesenchymal stem cells (MSCs), provide an attractive alternative to chondrocytes with regard to cell-based approaches to cartilage repair [1]. With the use of the proper three-dimensional serum-free culture conditions, expanded MSCs can be stimulated to differentiate along the chondrogenic pathway when the appropriate factors, such as certain members of the transforming growth factor (TGF)-β superfamily, are present [2–4]. This research has led to the first clinical application of autologous bone marrow stromal cells for the repair of full-thickness articular cartilage defects in humans [5, 6]. However, to date, the delivery of MSCs into cartilaginous lesions has neither clinically nor experimentally resulted in sustained regeneration of hyaline cartilage in vivo [7]. Inadequate delivery of the soluble factors necessary to drive the chondrogenic differentiation of the transplanted cells in vivo is a major impediment to effective chondrogenesis in situ [7]. To overcome this limitation, gene transfer approaches are being explored clinically [8] and experimentally [9–12] to enable the sustained delivery of chondrogenic and anti-inflammatory factors to cartilage defects.
Another obstacle was identified from studies of in vitro chondrogenesis using MSCs or chondrocytes treated with bone morphogenetic proteins (BMPs), members of the TGF-β superfamily. BMPs are a group of secreted polypeptides with pleiotropic roles in many different cell types and were originally identified by their ability to induce endochondral bone formation in ectopic extraskeletal sites in vivo [1, 7–10]. Among other BMPs, BMP-2 and BMP-7 are known to induce differentiation of mesenchymal progenitor cells and preosteoblasts into mature osteoblasts, and to enhance the differentiated function of osteoblasts, which have led to the clinical application of these proteins for bone regeneration [1, 7–10]. We and others have tested several BMPs for their potential use in cartilage regeneration including BMP-2, BMP-4, BMP-6 and BMP-7, which were shown to induce chondrogenic differentiation of mesenchymal progenitor cells and to up regulate the levels of type II collagen and aggrecan in chondrocytes and chondroprogenitor cells [1, 7–11]. During development of the limbs, however, BMPs along with other regulators also mediate the replacement of chondrogenesis by endochondral ossification comprising chondrocyte maturation, hypertrophy, transition from type II to type X collagen with subsequent chondrocyte apoptosis, while osteoprogenitor cells differentiate into osteoblasts and replace the cartilage with mineralized bone tissue. Equivalently, chondrogenic cultures induced by BMPs showed high expression of genes associated with chondrocyte hypertrophy, including collagen type (COL) X and indian hedgehog (IHH), among others [1, 7–11, 13]. This suggests that the chondrogenic differentiation of the MSCs advanced to the end stage, hypertrophic state that is typical of endochondral ossification during skeletal development. This conclusion correlates well with existing in vivo data. For example, delivery of BMP-2 expressing MSCs resulted in tissue hypertrophy and the formation of osteophytes, when transplanted orthotopically to osteochondral defects [14] or ectopically [15, 16] in small animal models. Moreover, such hypertrophy-associated changes are not exclusively found in terminal differentiated growth plate chondrocytes, but are also present in pathological conditions such as osteoarthritis [17, 18].
Inspired by these observations, we aim to further explore the effects of chondrogenic-induction by BMPs on hypertrophy, maturation and apoptosis. We have previously shown that adenoviral delivery of individual cDNAs encoding BMP-2 or TGF-β1 into primary MSCs is capable of driving chondrogenesis in culture [19, 20]. In the present study, using adenoviral-mediated gene transfer our aim was to compare the effects of BMP-4 and BMP-2 expression on chondrogenesis of primary MSCs and to investigate whether levels and extent of hypertrophy in vitro is influenced by the choice of transgene.
Materials and methods
Construction and preparation of recombinant adenoviral vectors
The complete coding sequence of the human BMP-4 gene [GenBank:M22490] cloned into λ gt10 bacteriophage vectors (ATCC No. 40342; Manassas, VA, USA) was isolated and purified according to standard protocols [21]. The isolated λ gt10 DNA was then digested with EcoRI to release the 1.7 kB sized BMP-4 cDNA insert, which was then cloned into the EcoRI site of the pAdlox shuttle vector, and first-generation, E1, E3-deleted, serotype 5 adenoviral vectors carrying the cDNAs for human BMP-4 were constructed by cre-lox recombination as previously described [22]. The vectors encoding BMP-2, firefly luciferase (Luc) or green fluorescent protein (GFP) from jellyfish were generated previously [22]. The resulting vectors were designated Ad.BMP-2, Ad.BMP-4, Ad.Luc and Ad.GFP, and suspensions of recombinant adenovirus were prepared by amplification in 293 cells followed by purification using three consecutive CsCl gradients [22]. Viral titers were estimated to be between 1012 and 1013 particles/mL by optical density at 260 nm and standard plaque assay.
Culture of human bone marrow-derived MSCs and adenoviral transduction
Bone marrow was harvested from the surgical waste of femurs of six patients, aged 48 to 63 years (mean age 55 years), undergoing total hip arthroplasty, after informed consent was given and as approved by the institutional review board of the University of Wuerzburg as described earlier [23]. The collected cells were spun at 1 × 103 rpm for five minutes, resuspended in complete DMEM (containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin), and plated at 4 to 6 × 107 nucleated cells per 75 cm2 flask (Falcon, Beckton Dickinson Labware, Franklin Lakes, NJ, USA). Unattached cells were removed after three days, and adherent colonies were cultured at 37°C, 5% CO2 in DMEM with 10% FBS supplemented with 1 ng/mL fibroblast growth factor (FGF) -2 for expansion of chondroprogenitor cells. Medium changes were performed every three to four days, and after 14 days adherent colonies were trypsinized and replated in several 75 cm2 tissue culture flasks. At confluence (approximately 1.2 × 106 cells/T-75 flask), the cultures were infected in 750 μL serum-free DMEM for two hours at a dose of 5 × 103 vp/cell of Ad.BMP-2, or Ad.BMP-4. Control cultures were similarly infected with Ad.GFP or Ad.Luc at 5 × 103 vp/cell, or remained uninfected. For comparison, an additional set of untransduced recombinant human protein controls were maintained, which were cultured in the presence of 10 ng/mL TGF-β1 protein, or 25 ng/mL BMP-2, or 25 ng/mL BMP-4 (all R&D Systems, Minneapolis, MN, USA). Following viral infection, the supernatant was aspirated and replaced with 10 mL complete DMEM.
Aggregate culture and transgene expression
Twenty-four hours post-infection, the MSC cultures were trypsinized, washed and placed in aggregate culture as described previously [24], and as modified by Penick and colleagues [25]. Briefly, MSCs were suspended to a concentration of 1 × 106 cell/mL in serum-free DMEM containing 1 mM pyruvate, 1% ITS + Premix (insulin, transferrin and selenous acid containing culture supplement), 37.5 mg/mL ascorbate-2-phosphate and 10-7 M dexamethasone (all Sigma, St. Louis, MO, USA), and 200 μL aliquots (2 × 105 cells) were distributed to a polypropylene, v-bottom 96-well plate (Corning, Corning, NY, USA) to promote aggregate formation. As mentioned above, to particular control aggregates 25 ng/mL BMP-2, 25 ng/mL BMP-4, or 10 ng/mL TGF-β1 recombinant protein (all R&D Systems, Minneapolis, MN, USA) was added to induce chondrogenesis. The cell pellets were cultured at 37°C, 5% CO2 and formed spherical aggregates within 24 hours. Changes of media were performed every two to three days, with the recombinant protein being also freshly added to the respective controls. The aggregates were harvested at various time points for further analyses.
Media conditioned by the aggregates over a 24-hour period were collected at day 3, 7, 14 and 21 of culture and assayed for BMP-2 and BMP-4 expression using the appropriate commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA).
Cell proliferation, glycosaminoglycan and alkaline phosphatase assays
For analysis of cell proliferation in aggregates, the WST1 test was performed at day 3, 7, 14 and 21 of culture according to the directions of the supplier (Boehringer, Ingelheim, Germany). Briefly, at time points indicated, pellets were washed twice with PBS and incubated with the WST1 reagent for two hours at 37°C. After this incubation, the formazan dye produced by metabolically active cells was quantified by measuring the absorbance at 450/690 nm in 96-well plates (Falcon).
Cell proliferation in aggregates was further assessed by quantitative detection of adenosine 5'-triphosphate (ATP), which correlates with the number of viable cells present using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Mannheim, Baden-Würtemberg, Germany) according to the manufacturer's instructions. Briefly, pellets were homogenized mechanically using a pellet pestle and mixed with 100 μL of CellTiter-Glo® reagent, which was generated by reconstitution of CellTiter-Glo® substrate with CellTiter-Glo® buffer. After incubation for 10 minutes at room temperature luminescence was measured using a plate-reading luminometer.
For analysis of glycosaminoglycan (GAG) content, aggregates were washed with PBS, digested with 200 μL of papain digest solution (1 μg/mL, Sigma, St. Louis, MO, USA), and incubated for 16 hours at 65°C. Total GAG content was measured by reaction with 1,9-dimethylmethylene blue using the Blyscan™ Sulfated Glycosaminoglycan Assay (Biocolor Ltd., Newtownabbey, Northern Ireland) as directed by the supplier. For normalization, DNA content of aggregates was also determined fluorometrically using the Quant-iT™ PicoGreen® kit as directed by the supplier (Invitrogen GmbH, Karlsruhe, Germany).
Alkaline phosphatase (ALP) activity was measured densitometrically using change in absorbance at 405 nm by the conversion of p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate, as described previously [26]. Briefly, aggregates were homogenized mechanically and incubated with 0.1 mL of alkaline lysis buffer (0.1 M glycin, 1% triton X-100, 1 mM MgCl2, 1 mM ZnCl2) at room temperature for one hour. Thereafter 100 μL of lysis buffer was added which was supplemented with p-nitrophenylphosphate (2 mg/mL; Sigma, St. Louis, MO, USA), and stopped after 15 minutes with 50 μL 50 mM NaOH before optical densities were determined at 405 nm in an ELISA reader. ALP activity was referred to a standard curve made from p-nitrophenol (Sigma, St. Louis, MO, USA), and normalized to the DNA content and given as relative ALP activity in U/μg.
Histological and immunohistochemical analyses
For histological analyses, aggregates were fixed in 4% paraformaldehyde for one hour before tissue processing. After dehydration in graded alcohols, the aggregates were paraffin embedded, and sectioned to 5 μm. Representative sections were stained using H&E for evaluation of cellularity and alcian blue (Sigma, St. Louis, MO, USA) for the detection of matrix proteoglycan. ALP activity was also detected by a histochemical assay performed according to the manufacturer's protocol (Sigma, St. Louis, MO, USA) and alternate sections were used for immunohistochemistry.
For immunohistochemical analyses, sections were washed for 20 minutes in Tris-buffered saline (TBS), and incubated in 5% BSA (Sigma, St. Louis, MO, USA). Following washing in TBS, sections were pre-digested with pepsin at 1 mg/mL in Tris-HCl (pH 2.0) for 15 minutes at room temperature for COL II detection, or with chondroitinase ABC (Sigma, St. Louis, MO, USA) for 10 minutes for chondroitin-4-sulfate (CS4) detection (5 U/mL in distilled water), or with 0.25% trypsin containing 1 mM EDTA for 15 minutes at 37°C for COL X detection, before sections were incubated overnight at 4°C primary antibodies diluted in 0.5% BSA. As primary antibodies monoclonal anti-COL II (Acris Antibodies GmbH, Hiddenhausen, Germany), anti-CS4 (Millipore GmbH, Schwalbach, Germany) or anti-COL X antibodies (Calbiochem, Bad Soden, Germany) were used. Immunostaining was visualized by treatment with peroxidase-conjugated antibodies (Dako, Hamburg, Germany) followed by diaminobenzidine staining (DAB kit; Sigma, St. Louis, MO, USA). The slides were finally counterstained with hemalaun (Merck, Darmstadt, Germany). For all immunohistochemical analyses, controls with non-immune immunoglobulin (Ig) G (Sigma, St. Louis, MO, USA) instead of the primary antibodies were performed.
Although more sophisticated and accurate methods of lacunae size determination have been described [27], we used a simple random field histomorphometric cell surface area measurement procedure to approximate cell sizes in aggregates. For each aggregate analyzed, three individual mid-sections stained with H&E or alcian blue were taken, and the surface areas of 10 randomly chosen lacunae by two independent investigators (AFS and BP) in a blinded fashion were measured from each of three representative microscope views taken from the center or the periphery (outer 200 μm area) section using the KS 400® computerized image analysis system (Carl Zeiss GmbH, Jena, Germany). At least three different aggregates per group and bone marrow preparations from five different preparations were analyzed.
For comparison, we also analyzed the sizes of the lacunae within different zones of growth plate cartilage obtained from a four-year-old child, from whom a sixth toe was removed. Specifically, from the toe we obtained four physes (two joints) and at least three sections per physis were analyzed by measuring the surface areas of 10 randomly chosen lacunae from each of three representative microscope views by two independent investigators (AFS and BP). The lacunae were taken from the reserve, proliferative, hypertrophic or calcifying zone.
Cell viability and apoptosis assay
As annexin 5 (Ann5) is expressed by hypertrophic chondrocytes and in osteoarthritic cartilage [17], we were next interested in the appearance of live and apoptotic cells within our aggregate system after 10 and 21 days, which was visualized using the Ann5-Cy3 apoptosis detection kit (APOAC; Sigma, St. Louis, MO, USA) as directed by the supplier. The assay uses the Cy3.18 dye as red fluorochome conjugated with Ann5-Cy3 for apoptosis detection through binding to phosphatidylserine epitopes on the plasma membrane of early apoptotic cells, and the hydrolysis of the non-fluorescent 6-carboxyfluorescein diacetate (6-CFDA) to the green fluorescent compound 6-carboxyfluorescein by the esterases of living cells to label viable cells. This combination allows the differentiation among early apoptotic cells (Ann5 positive, 6-CFDA positive), necrotic cells (Ann5 positive, 6-CFDA negative), and viable cells (Ann5 negative, 6-CFDA positive). Aggregates were incubated with 50 μL of the double labelling staining solution for 10 minutes at room temperature. After staining, aggregates were washed five times with 100 μL of binding buffer, fixed overnight in PBS-buffered 4% paraformaldehyde, dehydrated, infiltrated with isoamylacetate (Merck, Hohenbrunn, Germany), embedded in paraffin, and sectioned to 4 μm. Viable and non-viable cells were observed on the respective mid-sections using a fluorescence microscope and the appropriate green and red filters.
Total RNA extraction, semi-quantitative and real-time RT-PCR
RNA was extracted from MSC aggregates at the indicated time-points. For this, 6 to 10 pellets per group and time point for each donor were pooled and homogenized using a pellet pestle and repeated titration in 1 mL of Trizol reagent (Invitrogen, Karlsruhe, Germany). Total RNA was subsequently extracted using Trizol reagent with an additional purification step using separation columns (NucleoSpin RNA II kit; Macherey-Nagel GmbH, Düren, Germany) including a DNase treatment step according to the manufacturer's instructions. RNA from aggregates of each condition (2 μg each group) was used for random hexamer primed cDNA synthesis using BioScript reverse transcriptase (Bioline GmbH, Luckenwalde, Germany).
Primer sequences and product sizes, for semi-quantitative and real-time RT-PCR
RT-PCR primer sequences (5'-3')
Annealing temp. (°C)
Product size (bp)
Chondrogenic markers
Sense: TTTCCCAGGTCAAGATGGTC
Antisense: CTTCAGCACCTGTC CACCA
Sense: TGAGGAGGGCTGGAACAAGTACC
Antisense: GGAGGTGGTAATTGCAGGGAACA
Sense: CAGGACGACTTTGATGCAGA
Antisense: AAGCTGGAGCTGTCTGGTA
Sense: CTTACCCCTATGGGGTGGAT
Antisense: GTACATGGCCGTGAGGAAGT
Sense: ATCTGAAGAAGGAGAGCGAG
Antisense: TCAGAAGTCTCCAGAGCTTG
SOX9 (rt)
Sense: GGA GTGGAAGTTACTGACTGATG
Antisense: AGGCGTTTTGCTTCGTCAATG
Hypertrophy and osteogenic markers
Sense: GGACACAATGGATTGCAAGG
Antisense: TAACCACTGCTCCACTCTGG
Sense: CCCTTTTTGCTGCTAGTATCC
Antisense: CTGTTGTCCAGGTTTTCCTGGCAC
Sense: ACGCCGACCAAGGAAAACTC
Antisense: GTCCATAAACCACACTATCACCTCG
ALP (rt)
Sense: TGGAGCTTCAGAAGCTCAACACCA
Antisense: ATCTCGTTGTCTGAGTACCAGTCC
Sense: GAGGAGTCCCTGCATTATGA
Antisense: CAGGAAAATGAGCACATCGC
Sense: ACAGATGATGACACTGCCACC
Antisense: CATAGTAGAGATATGGAGTGCTGC
Internal control
Sense: AGGTGATTATCCTGAACCATCC
Antisense: AAAGGTGGATAGTCTGAGAAGC
For a more detailed mRNA expression profile of chondrogenic and hypertrophy associated genes, genetically-modified MSC aggregates were subjected to real-time quantitative PCR analyses. One microliter of each cDNA was used as template for amplification in a 50 μL reaction volume using BioTaq DNA Polymerase Taq (Bioline GmbH, Luckenwalde, Germany) and 50 pmol of gene-specific primers was used for COL II, SOX9, ALP and COL X as listed in Table 1. Real-time PCR conditions were as follows: 30 seconds at 94°C, 30 seconds at annealing temperature, 60 seconds at 72°C (see Table 1 for PCR conditions). Real-time PCR was performed with the DNA Engine Opticon system (MJ Research, Waltham, MA, USA) using SYBR Green (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) as fluorescent dye allowing determination of the threshold cycle at which exponential amplification of PCR products begins. Specificities of amplicons were confirmed by melting curve analyses by gel electrophoresis of test PCR reactions. For quantification mRNA expression was normalized to the expression levels of the housekeeping gene EF1α and relative expression levels compared with values from undifferentiated monolayer MSCs are shown using the relative expression software tool (REST) [28]. Each PCR was performed in triplicate on three separate bone marrow preparations for each independent experiment.
Statistical analysis
The data from the ELISA, WST1, ATP, GAG, DNA, and ALP content, cell surface area and RT-PCR analyses were expressed as mean values ± standard deviation (SD). Each experiment was performed in quadruplicate (n = 4) and repeated on at least three and up to six individual marrow preparations from different patients (m = 3 to 6), as indicated in the respective experiments. All numerical data were subjected to variance analysis (one or two factor analysis of variance) and statistical significance was determined by student's t-test, and level of P < 0.05 was considered significant.
Transgene expression by aggregates of genetically modified MSCs
Cell proliferation, GAG content and ALP activity
As primary MSCs were shown to be capable of expressing the BMP-2 or the BMP-4 transgene in aggregate culture, we examined the effects of BMP-2 and BMP-4 gene delivery on cell proliferation using the WST1 cell proliferation assay. At day 3 and 7 of culture the cell proliferation rate in MSC aggregates was approximately equal in all groups tested (Figure 1c). BMP-2 and BMP-4 transduced MSC aggregates maintained their proliferation rate over 21 days while Ad.GFP cells (Figure 1c) and unmodified control cultures (not shown) decreased rate of proliferation (Figure 1c). The same pattern was observed using the ATP test, where sustained high cell proliferation rates were observed at day 14 and 21 in BMP-2- and BMP-4-modified aggregates compared with the controls, while at the same time points, levels in the BMP-2-modified aggregates were significantly elevated compared with the BMP-4 cultures (Figure 1d). To quantitatively compare extracellular matrix synthesis among treatment groups, GAG levels in the aggregates after 21 days in culture were determined (Figure 1e). All aggregates infected with Ad.BMP-2 or Ad.BMP-4 showed significantly increased GAG production relative to those receiving Ad.GFP (Figure 1e), Ad.Luc or untransduced aggregates (not shown), which showed no evidence of chondrogenesis. At days 14 and 21, significantly elevated levels of GAG synthesis in the BMP-2 compared with the BMP-4 transduced cultures became apparent (Figure 1e). Indicative of hypertrophic chondrocytes we analyzed ALP activity, which was found to be significantly elevated at all time points in the BMP-2-modified aggregates compared with the GFP controls and BMP-4 transduced cultures, whereas significantly higher values in the BMP-4 modified cultures compared with the GFP controls could only be resolved at day 14 and 21 (Figure 1f).
Histological and immunohistochemical analyses of chondrogenesis
Uninfected aggregates maintained in the presence of recombinant BMP-2, BMP-4, or TGF-β1 protein were also chondrogenic as evidenced by lacunae formation, positive staining for alcian blue (Figure 2d), COL II and CS4 (not shown), although the stage of chondrogenesis seemed less progressed compared with that in the aggregates genetically modified with BMP-2 or BMP-4 (Figures 2b, c) after 21 days, while control cultures where growth factor supplementation was absent were non-chondrogenic.
Hypertrophic differentiation and apoptosis
A similar pattern of hypertrophy and apoptosis was observed in the untransduced control aggregates that were maintained in the presence or absence of recombinant BMP-2, BMP-4 or TGF-β1 protein (not shown).
Comparison of BMP-2 and BMP-4 modified MSC aggregates with immature growth plate chondrocytes
In the different types of aggregates examined in Figures 2 to 5, different cell morphologies were apparent, especially with respect to incidence and extent of lacunae formation. Thus we were next interested to know if it was possible to distinguish the types of aggregates produced by measuring the sizes of the respective lacunae, approximated by simple histomorphometric cell surface area measurement on aggregate sections. For comparison, we first analyzed the sizes of the lacunae within different zones of growth plate cartilage obtained from a four-year-old child, from whom a sixth toe was removed. These measurements were compared with those of the lacunae found in the center and periphery of the different treatment groups of genetically modified aggregates.
In contrast the GFP-modified aggregates showed no lacuna formation, either in the center or in the periphery of the pellets (Figure 6c). However the BMP-2- and BMP-4-modified aggregates displayed a chondrogenic phenotype with lacunae formation throughout the aggregates (Figures 6d, e). Analyses of cell surface areas in the different aggregate types revealed a mean value of 60.6 ± 14.5 μm2 in the center and 57.3 ± 12.4 μm2 the periphery of the Ad.GFP transduced aggregates, which showed no lacunae formation, of 541.3 ± 166.3 μm2 in the center and 386.1 ± 108.7 μm2 the periphery of the Ad.BMP-2 transduced aggregates, and of 307.8 ± 75.6 μm2 in the center and 248.7 ± 65.4 μm2 the periphery of the Ad.BMP-4 transduced aggregates (Figure 6f). Thus lacunae formed in both the BMP-2 and BMP-4 transduced pellets and led to significantly larger cell surface areas compared with the non-chondrogenic controls. Nevertheless, the lacunae formed in the presence of BMP-2 were larger than those formed by BMP-4 and approximated the size of lacunae noted in the calcifying zone of the human growth plate. In contrast, the lacunae that formed in the presence of BMP-4 were closer in size to those of the hypertrophic zone (Figures 6e, f).
Time course of chondrocytic and hypertrophic marker gene expression
Evidence of chondrocyte hypertrophy at the mRNA level in the BMP-2- and BMP-4-modified aggregates was reflected by a subsequent upregulation of COL X and OP at day 3, IHH and ALP at day 7 and RUNX2 at day 14 compared with Ad.GFP controls (Figure 7). These results suggest that BMP-2 and BMP-4 gene transfer induced a significant chondrogenic and hypertrophic response in MSC aggregates on mRNA level over time.
We and others have shown previously that primary MSCs undergo chondrogenesis following genetic modification with Ad.BMP-2 or Ad.TGF-β1 in aggregate culture in vitro [30–32] or when transplanted into chondral defects in vivo [14]. In the present study we adapted the MSC aggregate culture system to determine whether adenoviral delivery of BMP-4 can lead to chondrogenesis of primary MSCs in vitro, and to evaluate the extent of hypertrophy compared with BMP-2-modified cultures.
Adenoviral delivery of BMP-4 led to reliable chondrogenesis in human MSC aggregate cultures in a fashion comparable with that noted when the same dose of the BMP-2 transgene was administered as shown by staining with alcian blue, COL II and CS4 and the quantitative GAG assay, indicating increased GAG levels at days 14 and 21 in the BMP-2-modified aggregates. Notably, chondrogenic differentiation induced by either transgene increase levels of metabolic activity and cell proliferation compared with controls as evidenced by the WST1 and ATP assays. Moreover, high levels of chondrocyte hypertrophy occurred in MSC pellet cultures modified with either BMP transgene, as assessed by lacunar size, and expression of ALP, COL X and Ann5, and was overall slightly more advanced in the BMP-2-modified cultures compared with the BMP-4 modified cultures reaching significance levels in the ALP assay at all time points. Notably, exact the lacunar size comparisons between growth plate tissues and in vitro cell pellets might be inaccurate (Figure 6) due to artifacts that may appear during fixation and processing of these different types of tissues.
The RT-PCR data are in general agreement with the biochemical and histological observations, showing high levels of chondrogenic mRNAs in aggregates after BMP-stimulation, such as AGC, COMP, COL II, SOX9 and FMD. Likewise, transcripts encoding the hypertrophy associated genes COL X, OP, ALP, RUNX2 and IHH were also strongly present in both types of BMP-modified aggregates compared with controls.
These observations are in broad agreement with our previous study using alginate cultures of the murine mesenchymal C3H10T1/2 cell line, stably transfected with BMP-2 or BMP-4 cDNAs, where similar differences in the pattern of chondrogenesis and hypertrophy were observed [33]. Although in this previous study the expression of osteogenic and hypertrophy markers were partly attributed to the presence of β-glycerophosphate, similar increases in hypertrophy associated genes were seen in the present study where β-glycerophosphate was absent. Our results are consistent with those reported by Mackay and colleagues [34] and Mueller and Tuan [35] who likewise showed that the addition of β-glycerophosphate is not necessary to obtain a hypertrophic chondrocyte phenotype.
Our study is also in agreement with studies of in vitro chondrogenesis with primary MSCs using recombinant proteins, where BMP-4 was identified as a strong inducer of chondrogenesis [36], which produced less hypertrophy compared with BMP-2 [37]. Correspondingly, in vivo implantation of BMP-4 into abdominal muscles of rats led to ectopic cartilage and bone formation when delivered as recombinant protein [38] or via genetically modified cells [39]. Notably, the latter study revealed differential effects on chondrogenesis and osteogenesis depending on the type of cell analyzed [39]. Our study is limited to the use of bone marrow-derived MSCs and other effects may be seen when different cells are employed. Orthotopic BMP-4 gene delivery via retrovirus transduction of muscle-derived stem cells was shown to improve cartilage repair in rat osteochondral defects [40] and also when it was administered via adenovirus to dedifferentiated chondrocytes in osteochondral defects in rabbits [41]. In both studies improved repair in the BMP-4-treated defects compared with non-chondrogenic controls at 12 or 24 weeks respectively was observed, but detailed analyses of hypertrophy and apoptosis have not been performed [40, 41].
BMP-2 and BMP-4 have been implicated in embryogenesis and morphogenesis of various tissues and organs, where they regulate growth, differentiation, chemotaxis and apoptosis of a variety of cell types, including mesenchymal, epithelial, hematopoietic and neuronal cells [42]. Interestingly, in conditional knock-out experiments it has been found that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations failed to form in limbs deficient in both BMP-2 and BMP-4 [43]. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP-2 and BMP-4 [43]. In contrast, it was found that the loss of both BMP-2 and BMP-4 results in a severe impairment of osteogenesis. Deletion of BMP-4 alone did not impair osteogenesis or fracture repair, while deletion of BMP-2 alone did not impair osteogenesis but strongly prevented fracture repair [43–45]. This indicates that the presence of BMP-2 or BMP-4 is a prerequisite for osteoblastogenesis and these morphogens can apparently compensate for each other to a certain extent. However, they are less important for chondrogenesis [43–45].
During limb development, cartilage is gradually replaced by endochondral ossification, a process in which the chondrocytes mature, hypertrophy and express COL X with reduced production of COL II. Subsequently the cartilage becomes vascularized and infiltrated by osteoprogenitor cells, while the chondrocytes undergo apoptosis. The osteoprogenitor cells differentiate into osteoblasts, replacing the cartilage with mineralized bone; BMP-2 and BMP-4 are important regulators of these processes [46–48]. By using chondroprogenitor cells in high density, three-dimensional cultures these regulatory mechanisms can be partially recapitulated. Thus it is not surprising that studies on in vitro chondrogenesis using MSCs or chondrocytes incubated with members of the TGF-β superfamily reveal considerable hypertrophy and high levels of COL X expression. Although the use of COL X as a marker of chondrogenic hypertrophy in MSC-based systems has been questioned [13], it correlates well thus far to the existing in vivo data. For example, MSCs genetically modified to express BMP-2 display a significant level of tissue hypertrophy and osteophyte formation, when transplanted orthotopically to osteochondral defects [14] or ectopically [15, 49] in small animal models. TGF-β1 has been shown to induce hypertrophic and osteometaplastic changes in the synovium of rabbit joints, when directly delivered by first-generation adenovirus [50]. Furthermore, implantation of chondrocytes genetically modified to express BMP-7 has been shown to generate good hyaline cartilage repair tissue after six weeks in vivo, but after one year the repair cartilage is no better than that of controls, with only 0 to 28% of the transplanted cells being detectable at that time point [51]. This is agreement with a recent large animal study in pigs, that showed good hyaline cartilage repair after six weeks, when chondral defects were filled with periosteum cells genetically modified with BMP-2, while at six months the hyaline repair tissue had almost completely vanished and was replaced by fibrocartilage [52]. These observations might be attributed to mechanisms of hypertrophic differentiation and subsequent apoptosis, although clarifying analyses in vivo have not been conducted thus far. However, the presence of Ann5-positive cells in our hypertrophic aggregates modified with BMP-2 or BMP-4 in vitro correspond with these data.
Our data suggest that the degree of hypertrophic differentiation can be modulated by the choice of morphogenetic stimulus, while still maintaining efficient chondrogenesis. This permits cautious optimism that it may prove possible ultimately to achieve effective regeneration of articular cartilage in the absence of hypertrophic differentiation. Hypertrophic differentiation of neo-cartilage tissue with subsequent apoptosis development is certainly an undesired effect in cartilage defects in vivo, because this would lead to loss of the transplanted repair cells with subsequent matrix degradation. However, the relevance of chondrogenic hypertrophy and apoptosis of human MSCs induced by TGF-β superfamily members for cartilage repair in vivo has to be considered still unclear to this end, because this study is limited by its in vitro nature. Therefore, clarifying in vivo experiments are necessary before such factors can be recommended for further clinical use.
Adenoviral BMP-4 gene transfer efficiently induces the chondrogenic differentiation of human primary MSCs as effectively as BMP-2 gene transfer. However, both transgenes induced high levels of chondrocyte hypertrophy after three weeks of in vitro culture. It remains to be seen, whether it may be possible to develop methods for allowing robust chondrogenesis while preventing hypertrophic differentiation using different genes or proteins, which would presumably improve the outcome of cell-based approaches to cartilage repair in vivo.
aggrecan core protein
alkaline phosphatase
- Ann:
adenosine 5 triphosphate
- Ad:
adenoviral vector
bone morphogenetic protein
bovine serum albumin
carboxyfluorescein diacetate
chondroitin sulphate
cartilage oligomeric matrix protein
Dulbecco's modified eagle media
- EF1α:
elongation factor 1α
enzyme linked immunosorbent assay
fetal bovine serum
fibroblast growth factor
green fluorescent protein
hematoxylin and eosin
- Ig:
indian hedgehog
- Luc:
mesenchymal stem cell
phosphate-buffered saline
polymerase chain reaction
runt-related transcription factor 2
standard deviation
SRY (sex determining region Y) - box9
Tris-buffered saline
transforming growth factor.
We are grateful to Nadja Karl, Viola Monz and Christa Amrehn for their excellent technical assistance. This work was supported in parts by grants AR48566 and AR50249 from to National Institute of Arthritis and Musculoskeletal and Skin Diseases to SCG and CHE, by grant STE 1051/2-1 from the Deutsche Forschungsgemeinschaft (DFG) to AFS and UN, and by grant D-23 to AFS and AR from the Interdisciplinary Center for Clinical Research (IZKF) Würzburg.
Authors' Affiliations
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text:
Arlington is the latest place to have its schools threatened by "creepy clown" social media accounts.
Hoax social media posts, often featuring images of sinister-looking clowns, have threatened schools across the country. Thus far the posts have led to numerous arrests of teens suspected of making the threats, but no reported violence.
Last night, two Instagram accounts — @virginiaclowns and @dmv_clowns — posted similar threats, warning of shootings at a number of area schools, including Kenmore, Gunston and Thomas Jefferson middle schools in Arlington.
The threats have prompted a stepped-up police presence at Arlington schools this morning.
"We are aware [of the threats] and were in contact with the Arlington Police Department staff last night when we saw the messages," Arlington Public Schools spokesman Frank Bellavia told ARLnow.com. "As a precaution, ACPD has had an increased presence at our schools this morning."
It's September — Bid an especially hot and sweaty August adieu, September is here. Get ready for kids going back to school, fall beer tastings, outdoor festivals, Pumpkin Spice Lattes and cooler weather. As a reminder, however: it's still summer until Sept. 22.
Author Talk at Kenmore — Best-selling author Ann Patchett will be discussing her new book Commonwealth, which is set in part in Arlington, at an event on Thursday, Sept. 15. The event, at the Kenmore Middle School auditorium, is open to the public, with RSVP; it's sponsored by One More Page Books and Arlington Public Library. [Eventbrite]
CEB CEO Stepping Down — Tom Monahan, the CEO of the publicly traded, Rosslyn-based firm CEB, is stepping down. The search is now on for a new chief executive for the 4,500-employee company, which will be moving to a gleaming new office tower after construction wraps up, likely in 2018. [Washington Business Journal]
Flickr pool photo by Erinn Shirley
Arlington County Police Department officers are set to bond with locals over free barbecue, games and activities during a summer block party next week.
The police department is scheduled to host a summer block party at Kenmore Middle School (200 S. Carlin Springs Road) on Saturday, Aug. 27, from 9 a.m. to 4 p.m.
"This free event provides residents an opportunity to get to know their neighbors and police officers while enjoying interactive stations, safety demonstrations, entertainment, food and much more," ACPD said in a press release.
Some of the events planned for this year's block party include a police cruiser expo, K9 demonstration and a "mini academy" where attendees can try their hand at being a cop.
The party will also have a moon bounce and a "kid zone" with badge-making, a "need for speed" police driving experience and an area where kids can try on police gear.
Additionally, the celebration will have "lots of sauce" from Rocklands Barbeque, according to an ACPD promotional video:
It's not just free food at the Summer Block Party, it's @rocklandsbbq! Join us August 27 at @APSKenmore from 9 – 4! pic.twitter.com/7XJQ06Eg7h
— ArlingtonCountyPD (@ArlingtonVaPD) August 17, 2016
Image via Arlington County
Arlington County Police Department officers are set to meet with locals during fun community gatherings at six locations across the county tomorrow night.
The planned events are held as part of National Night Out, a "community-building campaign that promotes police-community partnerships and neighborhood camaraderie," according to a flyer distributed by the police department.
National Night Out celebrations are a chance for police and members of the community to come together, usually over free food and activities.
National Night Out events will be held at the following locations:
- Arlington Forest (200 block of N. Galveston Street) at 7:30 p.m.
- Nauck Town Square (24th Road S. and S. Shirlington Road) from 6:00-8:00 p.m.
- Barcroft Community House (800 S. Buchanan Street) from 6:00-7:30 p.m.
- Farlington Villages Pool 2 (3045 S. Buchanan Street) from 5:00-7:00 p.m.
- Park Glen Condo Associations: (800 block of S. Arlington Mill Road) from 7:00-8:00 p.m.
- Whitefield Commons: (106 N. Thomas Street) from 5:30-7:30 p.m.
Image via ACPD National Night Out Flyer
The Arlington County Police Department's Third District Team will be holding a "Safety & Community Awareness" event next week.
Sgt Damon Washington, who first proposed the free event, said it was part of the department's effort "to be more proactive and reach out to the community."
From 9 a.m. to 4 p.m. next Saturday, July 18, at Kenmore Middle School (200 S Carlin Springs Road), residents will have access to services such as bike registration and free Vehicle Identification Number (VIN) etching for their cars to help prevent theft.
The schedule includes several games, among them a distracted driving golf cart track where participants attempt to navigate the course with "drunk goggles" to simulate varying levels of intoxication and common distractions such as cell phones.
Additionally, ACPD officers have organized "15 Minutes Behind the Badge," an interactive simulation in which participants will receive a crash course in police training, be outfitted in faux-gear and be dispatched to a staged call in the area.
There will also be a K-9 demonstration, child safety seat checks and, for the younger set of safety-inclined residents, a moon bounce.
Rocklands and Wegmans are providing free food and drink for attendees.
Washington says that more that 75 officers are expected to be in attendance, including some from other police departments.
Arlington Scores Lidl HQ — Arlington County will be the home of the new $77 million U.S. corporate headquarters of Lidl, a discount German grocery chain that's seeking to expand in the United States. The headquarters is expected to create 500 new jobs in Arlington and will anchor the National Gateway office development near Potomac Yard. "Lidl chose Arlington for its U.S. corporate headquarters because of our commitment to diversifying our economy, a terrific workforce, regional transit connections and access to a major airport," Arlington County Board Chair Mary Hynes said in a statement. [Arlington County]
Arlington Teen Advances in Singing Competition — Kenmore Middle School student Samantha Rios, who is competing on the Telemundo singing program "La Voz Kids," has advanced to the semifinals and is now being coached by Reggaeton musician Daddy Yankee. [Washington Post]
Young Republicans Blast Anti-Gun-Store Tactics — Opponents of a gun store that's trying to open in Cherrydale are urging their supporters to confront the owners of the store and the shopping center in which it's opening in person. That has Republicans crying foul. "Having exhausted reasonable avenues, the anti-gunners encourage their flock to harass property owner," said the Arlington Falls Church Young Republicans. [Twitter]
Immigration Center to Open in Crystal City — A planned immigration services center in Crystal City, which has been delayed due to legal wrangling over President Obama's executive action deferring the deportation of certain illegal immigrants, is now reportedly set to open soon. Hundreds of U.S. Citizenship and Immigration Services employees will be working out of the office building at 2200 Crystal Drive, processing immigration applications and petitions. [Breitbart]
Praise for 'Alice' Production — The Encore Stage and Studio production of "Alice in Wonderland" is garnering critical kudos for its star, Brandi Moore, a Harvard-bound Washington-Lee High School senior. The show wraps up its run this weekend at Thomas Jefferson Community Theatre at 125 S. Old Glebe Road. [InsideNova]
Flickr pool photo by Erinn Shirley
Samantha Rios is a 13-year-old eighth-grader at Kenmore Middle School in Arlington, and her incredible voice has earned her onto the Spanish-language version of NBC's "The Voice" reality show.
Samantha appeared on La Voz Kids in March and earned all three judges' approval for her rendition of Whitney Houston's "I Wanna Dance With Somebody." Tomorrow, she is flying to Miami for the next round of performances, according to her chorus teacher, Raifel Faison.
Faison has taught chorus at the school just off Route 50 near Arlington's western border since 2003, and he said Rios is one of the most talents students he's ever had. Since she came into his class as a sixth-grader, she has only improved.
"She's always had the power, always had a powerful voice," he told ARLnow.com this afternoon. "She's never been shy or lacked confidence in her voice, but her tone has grown tremendously since sixth grade, her musicianship has grown. She's learned so much about her voice, how to control it, and her vocal technique is definitely improving."
Rios did tell the three judges that she was from Washington, D.C., in her blind audition, but her family calls Arlington home. Faison said Rios isn't just a talented signer, she's a well-rounded person.
"She's a very sweet, loving, intelligent caring young lady," he said. "She's very supportive of her classmates, a hard worker. Whatever positive adjective you can think of would probably describe her."
Rios is on Team "Daddy Yankee" the stage name of one of the trio of judges, one of 10 team members. According to her bio on La Voz Kids' website, she's already sung for the president multiple times and is a member of a D.C. gospel choir.
La Voz Kids airs Sunday nights at 8:00 p.m. on Telemundo.
Photo via La Voz Kids
A pair of Yorktown High School students, and flying projectile enthusiasts, are hosting a two-day, free dodgeball tournament next weekend.
The tournament starts at 11:00 a.m. Saturday, April 25 and runs all day, both days, at Kenmore Middle School (200 S. Carlin Springs Road). Adults, teenagers and children are welcome, and will be split into divisions based on age. There are divisions for 11 and younger, 14 and under, 17 and under and 18 and over.
The tournament is the brainchild of Yorktown students Patrick Wallace and Hayden Kickbush. While the tournament is free, Wallace and Kickbush are asking for attendees to donate what they can to fund the tournament and raise money for physical education equipment for D.C. area schools in need. They have set up an online fundraising page, and have already raised $1,325 of a stated $9,000 goal.
The boys got the idea for the tournament from a trip to Hawaii, where one of them saw a community dodgeball tournament and was inspired, according to the tournament's website.
"We experienced a heartwarming community and a tournament based on good morals and having a good time," the website reads. "We were so moved by the experience that we wanted to replicate the same thing here in the Arlington area. Arlington Dodgeball aims to have a very community friendly tournament while also giving back to the community."
According to Wallace's father, Marc, the boys have received nonprofit approval by the IRS, so all donations are tax-deductible. The tournament itself will cost about $7,000 to run, Wallace said. If there is money left over, the boys will determine which school in the area will receive the donated equipment.
Registration for the tournament is limited and filling up fast, Wallace said, so individuals or teams should register as soon as possible to ensure a spot.
Photo via Arlington Dodgeball
A sixth-grader was attacked by two seventh-graders outside Kenmore Middle School last Thursday after school hours, and the incident has raised concerns among parents about how the school handles cases of bullying and violence.
According to Kenmore Principal John Word, a seventh-grader said the sixth-grade victim had called him "a racial slur" over the summer, and the seventh-grader and his friend waited until about 4:30 p.m. on Thursday to retaliate.
In the field between Kenmore and Carlin Springs Elementary School along S. Carlin Springs Road, the two seventh-graders hit the younger boy in the face at least twice, while a crowd of other students watched, school officials confirmed. The victim reportedly received bruises on his face but didn't need to receive medical treatment.
An administrator quickly broke up the fight, the school said, but police were called and filed a report. The boy's mother, who will not be named to protect the identity of the minor, said she did not receive any communication from the school until she went herself the following day.
The incident sparked concern among parents of Kenmore students, to the point where the school held a community meeting yesterday afternoon to address the attack.
"This was not random, it was targeted and wrong," Word told a group of more than a dozen parents in the school's library yesterday. "After interviewing those culprits, the victims and some witnesses, I was convinced that this incident should result in the most severe consequence I could administer."
The seventh-graders initially were given two-day suspensions, Word said, but he decided to increase their punishments after the school completed its investigation. Word could not reveal the seventh-graders' final punishment due to student confidentiality laws, but according to the APS Handbook, the most severe punishment allowed for incidents like "physical altercations, fighting and bullying" is "a maximum of ten (10) consecutive days out-of-school suspension, request for disciplinary hearing for additional suspension time and/or a recommendation for expulsion."
While Word said he waited to reach out to the community until he had all the facts, that explanation did not ease the concerns of the parents at yesterday's meeting.
"I'm concerned about my children's safety at this school," said a parent, who requested her name not be used due to potential "repercussions upon our children." "There was no message given to our kids… The bylaws show that you have 48 hours to respond. Now we have all these kids hearing these things [about the attack], and they wonder why no one has talked to them about it in school."
When the victim's mother began to introduce herself at the meeting, she couldn't finish her sentence before she began crying. She clutched a tissue for the majority of the hourlong gathering, while listening to the meeting's translation by a Spanish interpreter sitting next to her.
The assault — which is how the school classified the incident — took place exactly one week after a separate altercation at Gunston Middle School. ARLnow.com received a tip about a seventh-grader at Gunston who, his parents say, was "sucker-punched" in the hallway during school hours. The victim had received "verbal bullying" during class and "a substitute teacher did not intervene on his behalf," the parent wrote. (more…)
(Updated at 1:15 p.m.) The Arlington School Board is reconsidering the plan to build a 600-seat elementary school at the Kenmore Middle School site in Glencarlyn.
Despite voters approving $4.5 million in design costs for the school in a 2012 referendum, the Board is looking at diverting that investment to prepare for middle school overcrowding in the coming years, which is projected to be more serious than the capacity issues in elementary schools.
School Board Chair Abby Raphael, in a letter sent to parents and community members who have inquired about the issue, says its updated projections call for elementary schools in Arlington to be 3 percent over capacity in FY 2019, while middle schools are projected to be 16 percent over capacity in the same time period.
Raphael also referenced the objections from Glencarlyn residents from 2012 as a reason to re-evaluate building the school in the neighborhood, saying "the community raised significant concerns about the traffic and transportation issues" surrounding a new, 600-seat school in the area.
APS is revisiting the plan in advance of their next Capital Improvements Program for FY 2015-2024, which will be adopted in June. Raphael wrote that no decisions have been reached on what schools to build, if any, or if the School Board elects to construct additions onto existing schools.
Civic activist Monique O'Grady is trying to organize a campaign against the apparent backpedal. O'Grady said she's disappointed that APS is considering abandoning its plans.
"The numbers still show that south Arlington will face more than an elementary school's worth of overcrowding, so I believe the plan should move forward," she wrote in an email. "I believe middle school should be addressed, but it shouldn't come at a cost of 770 South Arlington elementary students being in trailers and with yet-to-be-mentioned programs being moved."
O'Grady said the school should still be built while APS comes up with creative, cost-effective solutions to address anticipated middle school overcrowding.
"I worry that increased development in South Arlington, especially of apartment buildings and condos, will result in more students than currently projected and that South Arlington schools will become even more crowded than anticipated," she said. "This is not a time to pull back from researched, planned and approved permanent elementary capacity in South Arlington. I think it is important for the South Arlington community to stand up and ask the school board not to turn South Arlington into a trailer park."
APS acknowledges it does not have the finances to build capacity to accommodate 100 percent of the projected growth. No matter what comes out of the CIP, trailers will still be used as classrooms. The elementary school in Glencarlyn was originally slated to open in 2017.
Rep. Jim Moran (D-Va.) and a group of like-minded panelists made the case for immigration reform Tuesday night at a community forum in Arlington.
Entitled "Building a Stronger Nation: Reforming Out Broken Immigration System," the Moran-organized forum attracted several dozen attendees to Kenmore Middle School's auditorium. The congressman and the panelists told the audience that immigration reform would energize the economy, bring in additional tax revenue, and enable immigrants to live a more productive and fulfilling life.
In his opening remarks, Moran said bipartisan immigration legislation that's currently being crafted in the Senate has a better shot at becoming law than any other recent attempt at immigration reform.
"The possibility for reform today may be better than it's ever been," he said. "Now is the best time in recent memory for enacting comprehensive immigration reform. But the enactment of reforms is by no means guaranteed… in a Congress that can't seem to agree on anything of consequence."
Moran said immigration reform is particularly important in Northern Virginia, where 27 percent of the population is foreign-born. (Of that foreign-born population, 38 percent of come from Latin America, 36 percent from Asia, 16 percent from Africa and 10 from Europe, according to statistics cited by Moran.)
Panelists made moral and economic arguments for immigration reform.
Patrick Oakford, who researches immigration issues for the liberal Center for American Progress, said that legalizing the 11 million undocumented immigrants in the United States could boost the economy by $832 billion over 10 years while raising the wages paid to immigrants.
Arlington County Board Chair Walter Tejada said immigration reform would help cash-strapped local governments. It would also help police departments, he said, by facilitating better cooperation with an immigrant community that's currently fearful of law enforcement.
"The future of our nation is brighter by providing a path for citizenship," Tejada said. "We really need to get behind and support our leaders in Congress."
Other panelists tried to shoot down some of the arguments against immigration reform.
Kristian Ramos of the New Policy Institute, pro-immigration think tank, said immigration reform won't open the floodgates to Mexican immigrants. He said that Mexico's growing economy has helped to significantly reduce the flow of undocumented immigrants into the United States by providing more jobs and opportunities in Mexico. He also pointed out that that crime is down near the U.S.-Mexico border.
Two months after holding a raucous forum on gun violence, Rep. Jim Moran (D-Va.) is planning a public forum on another hot-button topic.
On Tuesday, May 14, from 7:00 to 9:00 p.m. at Kenmore Middle School (200 S. Carlin Springs Road), Moran will host a forum entitled "Building a Stronger Nation: Reforming Our Broken Immigration System."
Just as the gun violence forum featured panelists that largely shared Moran's gun control views, the immigration forum will feature panelists who favor liberal immigration policies: County Board Chair Walter Tejada, plus representatives from the Center for American Progress, the National Immigration Law Center and the American Immigration Lawyers Association.
"The panel discussion will outline systemic problems in our current immigration system and layout the comprehensive reform plans that are currently under consideration in Congress," said a press release for the event.
"There are an estimated 10 – 11 million undocumented immigrants living in America, the majority having settled here more than a decade ago," the press release said. "Reforming the broken immigration system to resolve the status for these individuals has the potential to boost the entire U.S. economy, adding over $800 billion to the national GDP over the next decade and creating over 100,000 more jobs per year."
(Updated at 10:15 p.m.) Arlington County police are investigating a serious pedestrian accident on Carlin Springs Road, in front of Kenmore Middle School.
Around 7:00 p.m., a man in his 30s was struck by a car on Carlin Springs Road just south of Route 50, according to Arlington County Police Department spokesman Dustin Sternbeck. The man suffered "severe head trauma" and was transported in critical condition to Inova Fairfax Hospital.
The driver of the striking vehicle remained on scene, Sternbeck said. The driver was eventually transported to a local hospital for minor injuries caused by broken glass.
Carlin Springs Road was closed to traffic between Route 50 and 2nd Street while police investigated the accident. The ramp from eastbound Route 50 to Carlin Springs Road was also closed.
Firefighters were called to the scene to wash down a pool of blood where the man came to rest on the roadway.
Thirteen new Arlington County police officers were sworn in this afternoon at Kenmore Middle School.
The recruits — eight male, five female — pledged an oath and were given their badges. The ceremony followed the recruits' graduation from a regional police academy. They will now undergo nearly half a year of field training before becoming full officers.
The ceremony included a speech from Arlington County Police Chief M. Douglas Scott. The swearing in was conducted by Arlington Clerk of the Circuit Court Paul Ferguson. Loved ones of the new officers helped to pin the badges on their uniforms while other friends and family members in the audience applauded.
After the swearing in, the event continued with a ceremony for 12 police department members who have received promotions.
Also recognized was Officer Ronald Grannis, who received the Departmental Purple Star after being seriously injured in a crash with an impaired driver on July 20, 2011. Grannis "spent over a month in the hospital and has battled through over ten surgeries while continuing to fight to return to full duty," according to police.
The Glencarlyn Citizens' Association is asking the Arlington County Board for help in its fight against a new proposed school in the neighborhood.
To address school capacity issues, Arlington Public Schools is planning to build a number of new schools, including a new 600-seat "choice" elementary school on the site of the existing Kenmore Middle School/Carlin Springs Elementary School campus.
The Citizens' Association says the new school, slated to be built by 2017, would bring the total number of students attending schools in the Glencarlyn neighborhood to 2,600, including at Kenmore, Carlin Springs and nearby Campbell Elementary School. That, the association says, presents major traffic, parking and open space issues that will degrade the quality of life for residents.
The association is asking for the County Board's help after not getting a satisfactory response from the School Board.
"We have tried to raise our concerns with the School Board, but our community was not consulted during the planning process, despite our requests that it do so, nor has it been responsive to our questions and concerns," Glencarlyn Citizens' Association President Peter Olivere wrote in a letter to the County Board (after the jump). "We need your help."
Olivere told reporters that Glencarlyn residents do not want to be portrayed as having a "Not-In-My-Backyard" attitude.
"Please, we very much do not want to be categorized as NIMBY; we only want a process which fully addresses the community concerns before a final decision is made, which is the 'Arlington Way,'" he wrote.
As previously reported, Arlington Public Schools is facing a significant capacity crunch. The school system is expected to reach capacity at the elementary school level by next fall. The new choice school in Glencarlyn is one of five proposed new capacity-generating construction projects throughout the county.
The full letter from the Glencarlyn Citizens' Association, after the jump.

subset: commoncrawl


text:
You know what???
I have fallen in love with you for the eleventh time now…
God! Those tears of joy…
Those Hurrahs and cries.
The anticipation burning till the end,
And that look on your face not sure of a foe or a friend.
Man! I keep romancing with the TV
And hope that you realize me as I am you biggest devotee.
That double fault was just the cherry on the top,
Winner is the best man of all…
Oh critics! Just shut up!
You hurt my hero in the last slams.
The eerie 5-setter didn't trouble him at all
The thought of losing was all that conspired his fall.
But look at you RAFA!
Amongst the best you shined.
The kingdom of clay is again at your mercy
Grass and Hard Court all clap in glee.
I am at the foot of TV,
Waiting for the coming glory…
Many more slams to come
Many more titles to be won
Each time you pick your racquet
I am waiting for a winner from behind the baseline.
That aggressive look on your face
Strength in your forearm make your opponents fade.
Hope to see you once live
Be it Phillipe Chatrier, Centre Court, Rod Laver Arena or Arthur Ashe…
Continue being the conqueror of the world
I hope you conquer your illness along with all the titles.
See you back on the court with a bang
I am sure I will fall in love with you at every coming instant…
© Copyright 2017 GoodyBravo. All rights reserved.
Status: Finished
Genre: Poetry
Poem / Poetry
Poem / Poetry
Poem / Poetry
Paste the link to picture in the entry below:
Paste the link to Youtube video in the following entry:
Cannot annotate a non-flat selection. Make sure your selection starts and ends within the same node.
An annotation cannot contain another annotation.
There was an error uploading your file.


text:
--- In iris-photos@y..., Linda Mann <lmann@v...> wrote:
> Janet, the form and color pattern of your JET BLACK look identical
to my
> falsely identified 'CHERRY SMOKE' posted last spring. However, on
> monitor, your photo of JET BLACK looks bluer than my unknown. My
> unknown is closer to the color of DIABOLIQUE - definitely a red
> rather than a blue black. Do you have a SCHREINER'S catlog handy to
> color match your JET BLACK to something for comparison (assuming the
> colors of the printed photos came out looking the same in every
> catalog...)?
> Thanks..
> Linda Mann
Jet Black doesn't have a red glow, the photo is very true to colour.
For your unknown, check out Superstition, an old black that is
definatly a red-black.
Chuck Chapman, Guelph, Ontario, Canada
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text:
Cream Of Sorrel Soup
|Chicken broth
|8 Cup (128 tbs)
|3 Medium, peeled and diced
|1 1⁄2 Pound, well washed
|1 Cup (16 tbs)
|Chopped chives
|2 Tablespoon (For Garnish)
|To Taste
|To Taste
1.In a pan, pour chciken broth and cook until boil.
2.Put potatoe sin the pan and allow them to cook for 15 minutes.
3.Put sorrel in the pan and cook for 19 more minutes.
4.Once done, blend the ingredints well and heat again.
5.Put salt and pepper as per the taste.
6.Garnish with cream and serve hot in bowls.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size
Calories 271 Calories from Fat 44
% Daily Value*
Total Fat 5 g7.1%
Saturated Fat 1.4 g7%
Trans Fat 0 g
Cholesterol 2.7 mg0.9%
Sodium 946.6 mg39.4%
Total Carbohydrates 54 g18%
Dietary Fiber 8.3 g33.3%
Sugars 15.8 g
Protein 8 g15.7%
Vitamin A 24.7% Vitamin C 200.2%
Calcium 19.1% Iron 29.4%
*Based on a 2000 Calorie diet


text:
Composer of the Week
Each week the children of Ark Conway Primary Academy listen to music that ties in with their curriculum studies. The music is played around the school and the children can hear it as they move between classes. For each theme a key musician or composer is selected as an example and children are taught a little bit about the genre and/or artist in assemblies.
Leonard Cohen (w/c 21/11/2016)
Canadian singer-songwriter Leonard Cohen was born in 1934. An early writer and guitarist, Cohen began to compose and release folk-rock and pop songs by the mid-1960s. One of his most famous compositions is "Hallelujah," a song released on 1984's Various Positions. Cohen was inducted into the Rock and Roll Hall of Fame in 2008, and he received a Grammy Award for lifetime achievement in 2010. Cohen died in 2016 at the age of 82.
George Frideric Handel (w/c 31/10/2016)
Baroque composer George Frideric Handel was born in Halle, Germany, in 1685. In 1705 he made his debut as an opera composer with Almira. He produced several operas with the Royal Academy of Music in England before forming the New Royal Academy of Music in 1727. When Italian operas fell out of fashion, he started composing oratorios, including his most famous, Messiah. Handel died in London, England, in 1759.
Ray Charles (w/c 10/10/16)
Born in Georgia in 1930, Ray Charles was a legendary musician who pioneered the genre of soul music during the 1950s. Often called the "Father of Soul," Charles combined blues, gospel and jazz to create groundbreaking hits such as "Unchain My Heart," "Hit the Road Jack" and "Georgia on My Mind." He died in 2004, leaving a lasting impression on contemporary music.
Wolfgang Amadeus Mozart
Wolfgang Amadeus Mozart (27 January 1756 – 5 December 1791) was a prolific and influential composer of the Classical era. Born in Salzburg, Mozart showed prodigious ability from his earliest childhood. Already competent on keyboard and violin, he composed from the age of five and performed before European royalty.
At 17, Mozart was engaged as a musician at the Salzburg court, but grew restless and traveled in search of a better position. While visiting Vienna in 1781, he was dismissed from his Salzburg position. He chose to stay in the capital, where he achieved fame but little financial security. During his final years in Vienna, he composed many of his best-known symphonies, concertos, and operas, and portions of the Requiem, which was largely unfinished at the time of his death. The circumstances of his early death have been much mythologized. He was survived by his wife Constanze and two sons.
He composed more than 600 works, many acknowledged as pinnacles of symphonic, concertante, chamber, operatic, and choral music. He is among the most enduringly popular of classical composers, and his influence is profound on subsequent Western art music. Ludwig van Beethoven composed his own early works in the shadow of Mozart, and Joseph Haydn wrote that "posterity will not see such a talent again in 100 years"
Claude Debussey (Composer of the Week w/c 12/9/16)
Claude Debussy was born in St. Germain-en-Laye, near Paris, France. His father was a salesman and kept a china shop. His mother was a seamstress. Debussy's piano teacher, Mme. Maute, had been a student of Frédéric Chopin. She sent Debussy to the Paris Conservatory, where he studied from 1872-84 with César Franck, Ernest Guiraud and others. He lived at the castle of Nadezhda von Meck and taught her children. She was a wealthy patroness of Pyotr Ilyich Tchaikovsky, and eventually Debussy played all pieces by Tchaikovsky in addition to other classical repertoire. She also took Debussy on trips to Venice, Vienna and Moscow. In Vienna he heard "Tristan und Isolde" by Richard Wagner and later admitted that it had influenced him for a number of years. Debussy won the Prix de Rome twice--in 1883 and 1884--and the money covered his studies at the Villa de Medici in Rome for the next four years. In Rome he met Franz Liszt and Giuseppe Verdi and heard more of Wagner's music, which made a strong impression on him.
Debussy became influenced by the impressionist poets and artists in the circle of Stéphane Mallarmé. In 1890 he wrote his most famous music collection for piano, "Suite bergamasque", containing "Clair de Lune". His "Prelude to the Afternoon of a Faun" continued the most productive 20-year period in his life. He composed orchestral "Nocturnes", "La Mer", "Images" , and the intricate ballet "Jeux" for "Ballets Russes" of Sergei Diaghilev. In 1908 Debussy married singer Emma Bardac after they had a daughter, Claude-Emma. Debussy called her Chou-Chou and composed for her the collection of piano pieces "Children's Corner Suite". The beginning of WW I and the onset of cancer depressed Debussy. He left unfinished opera, ballets and two pieces after stories by Edgar Allan Poe that later were completed by his assistants. He died on March 25, 1918, in Paris.
Danny Elfman
Danny Elfman was born in 1953 in Los Angeles, California, where he currently resides. Over the last 20 years, he has established himself as one of Hollywood's leading film composers. Elfman has written close to 50 film scores featuring his unique sound, including Batman, Spider-man, Men in Black, Beetlejuice, Edward Scissorhands, The Nightmare Before Christmas, and Pee Wee's Big Adventure. In addition to these signature soundtracks, he has scored such diverse films as Big Fish, Good Will Hunting, Dolores Claiborne, Midnight Run, To Die For, Dead Presidents, Sommersby, and Chicago. For television, Elfman created the infectious themes to The Simpsons and Desperate Housewives. His honors include a Grammy and three Academy Award nominations.
Arvot Part
The composer of the week is Arvo Pärt (born 11 September 1935) who is an Estonian composer of classical and sacred music. Since the late 1970s, Pärt has worked in a minimalist style that employs his self-invented compositional technique, tintinnabuli. His music is in part inspired by Gregorian chant. Pärt has been the most performed living composer in the world for 5 consecutive years.
Black History Month
Out of respect for Black History Month, Ark Conway students listened to Ray Charles and Louis Armstrong in October 2015. The two performers span over several genres. Ray Charles was famous for combining Blues, Gospel and Rythm & Blues to create a soulful sound. He is said to be the pioneer of Soul music. Louis Armstrong or Satchmo as he was also known was a great Jazz Musician. He came into public knowledge in the 1920's, playing trumpet. He soon made it as a vocal artist and was one of the foundational members of the jazz movement. Both men pioneered their own genre of music, they not only integrated sounds to create a unique musical experience, they also helped integrate society. Their music crossed over the racial borders that were prominent in American society in the early part of the 20th Century, and posibly helped to break down some of the tensions.
|Where you can listen
|Rythm & Blues, Blues, Soul, Gospel
|Ray Charles
|Jazz, Easy Listening
|Lois Armstrong
|Avant-Garde Classical
|Arvo Part
|Film Scores, Easy Listening
Soul, Blues, Jazz
|Danny Elfman
Ray Charles


text:
more Topics
National accounts figures may signal recession
KERRY O'BRIEN: On Wednesday, the national accounts for the last quarter of the year 2000 will be released.
That is, the calendar year.
They may show negative growth -- that is the first significant step down the road to recession.
At best, they'll show a dramatic slowdown.
There are other gloomy signs -- building activity sharply down, job ads sharply down, business investment down.
For a government that hoped to go to the election later this year, contrasting its successful economic management with Labor's failures, none of that is good news.
And John Howard's decision to bow to public pressure and cut the fuel excise has left him with very little room to manoeuvre in his budget, if he's not to blow the surplus and what remains of his reputation as a prudent manager.
Here's political editor Fran Kelly.
FRAN KELLY: The Prime Minister began this election year on the theme of economic management, contrasting his, with Labor's.
JOHN HOWARD, PRIME MINISTER: In the end, Kerry, the people of Australia have to choose between this Government and the economic strength and stability it has given Australia and a return to the 17 per cent interest rates, the $80 billion of Government debt and the 11 per cent unemployment which is a hallmark of the last time Mr Beazley had a senior position in Government.
FRAN KELLY: Yesterday John Howard was still claiming credit for strong economic credentials, but conceding the gloss is coming off the economy, forecasting a poor growth figure when the national accounts come down on Wednesday.
JOHN HOWARD: They'll be lower than previous ones.
I don't know about whether something will be negative or not.
I just don't know.
I can't tell at this stage.
But, it does look as though they will be lower than what they have been.
FRAN KELLY: Low growth.
And that follows figures out last week showing a fall in housing and business investment.
There were more bad figures today, though the Treasurer preferred to dwell on the increase in retail spending as a sign that all is not ill in the post-GST economy.
The trouble is that while some of us are spending more in the shops, a slowing economy means more of us will end up out of work.
And while Peter Costello was once tipping an unemployment rate of around 5 per cent, today he was conceding that holy Grail is now a more distant dream.
PETER COSTELLO, TREASURER: It's important that we continue to run a strong economy and if you continue to grow the economy at 4 per cent, you would continue to eat into the unemployment rate at about O.5 per cent per annum, which is what happened over the last four years.
FRAN KELLY: More bad news on the economic front today, pointing directly to rising unemployment.
The ANZ job ads slumped 10 per cent, a dramatic monthly fall, considering there were no factors to take into account, like the Olympics or Christmas.
SAUL ESLAKE, CHIEF ECONOMIST, ANZ BANK: The only two occasions that have produced larger declines than this, without it being for those reasons, were in the 1982-83 recession.
The decline in job ads over the past 12 months of nearly 28 per cent, is the largest decline over a single year, since the recession of the early 1990s.
FRAN KELLY: That word recession is striking fear into the heart of the Government.
And striking directly at its claims of strong economic management.
It takes two quarters of negative growth to make a recession but one takes us halfway there.
Labor's been saying for some time that the current slowdown is due to the GST rather than external factors, and that's a claim that the housing industry for one, won't disagree with.
RON SILBERBERG, HOUSING INDUSTRY ASSOCIATION: We'd like to believe that the worst is over for the home building industry.
But when you consider that activity has dropped by about 50 per cent on the same time last year, you are on the canvas, you're down to the count of eight, there's a reasonable chance that the figures -- the next round of figures will be a little better -- but there's a long way to go.
We're not out of the woods yet.
FRAN KELLY: Last week, Housing Industry leaders told the Prime Minister the GST slowdown in their sector alone, would wipe almost 1 per cent off GDP.
ANZ Chief Economist Saul Eslake, doesn't agree entirely with Labor or the housing industry but he does agree the GST has been a contributor to the slowdown.
SAUL ESLAKE: It's been a contributor but not the sole factor in the slowdown that next Wednesday's national accounts will reveal.
It's certainly has been a major influence on the slump in housing activity, which in turn has been a major reason for the decline in the growth rate since the middle of last year.
On the other hand, the income tax cuts have probably helped to support consumer spending on a stronger plane than otherwise would have been the case.
FRAN KELLY: And guess where the GST-induced slump in the housing industry has hit hardest since July of last year?
RON SILBERBERG: Well, in Queensland over the past six months, five building businesses have stopped building every day.
Five a day.
FRAN KELLY: And that's very bad news for a Government heading into a critical by-election in that State, in under two weeks time.
If Ron Silberberg is right, small business in Queensland could well be in a blaming mood by then.
Another negative for the Ryan by-election could be the weekend fracturing of the state Coalition in Queensland.
The Prime Minister has warned that disunity is death and any hint between a split between the Nationals and the Liberals at a State level, being repeated Federally would certainly be fatal, a disaster Queensland Federal MPs were quick to rule out today.
DE-ANNE KELLY, NATIONAL PARTY BACKBENCHER: Look, it will be a distraction, there's no doubt about that.
But the Federal Coalition is secure.
Nobody, including the Queensland National Party, is going to hand Government at the next Federal election to the Labor Party and Mr Beazley.
So you know, the Federal Coalition is very secure.
We're not about helping Labor Party to win Government.
FRAN KELLY: But how much help will Labor need to win Ryan?
The Greens are already offering to help with preferences for Labor, if Kim Beazley comes up with a public pledge to end the rapid rate of land clearing in that State.
And that leaves John Howard.
He is hoping the Reserve Bank will come to his rescue announcing another interest rate cut on Wednesday.
After all, there's nothing like a rate cut to drown out pesky talk of negative growth and recessions.
And to give the Government another platform to start talking up their strong economic credentials.
Transcripts on this website are created by an independent transcription service. The ABC does not warrant the accuracy of the transcripts.
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text:
Special equipment: Muffin tin with paper muffin cases
Preheat the oven to 400 degrees F.
Put the flour, baking powder, baking soda, cocoa, sugar, and 3/4 cup of the chocolate chips into a large bowl. Pour all the liquid ingredients into a measuring jug. Mix the dry and wet ingredients together, remembering that a lumpy batter makes the best muffins. Spoon into the prepared muffin cases. Sprinkle the remaining 1/4 cup chocolate chips on top and then bake for 20 minutes or until the muffins are dark, risen and springy.
Copyright 2004, Feast, Hyperion, All Rights Reserved
The best comfort food is comforting both to cook and to eat, like Ree's pork chops.


text:
TripAdvisor ratingRead reviews
Hotel Capacity:Total Number of Rooms - 21
Novotel Singapore Clarke Quay
The elegant Novotel Clarke Quay enjoys a great location just next to Singapore River, with wonderful views over the city and Marina Bay. Fort Canning Park is just a stone's throw away, while other attractions such as Central Mall, Chinatown or the famous shopping paradise Orchard Road are within walking distance. Suntec City Convention Centre is within easy reach; the Clarke Quay MRT Station can be reached within a short stroll, offering easy access to all areas of the city.
The hotel offers spacious and well-equipped rooms in a contemporary design. Business guests will appreciate WIFI, the business centre as well as the excellent conference facilities. Other first-class services and amenities include an outdoor pool, a concierge service, a bar and three restaurants serving local and international cuisine. This hotel is a perfect choice for business travellers and holidaymakers alike.
Accommodation details
Address:177A RIVER VALLEY ROAD, CITY CENTRE
General facilities
- Laundry/Valet service
- Exercise gym
- Internet services
- Currency exchange
- Wireless internet connection in public areas
- Poolside snack bar
- Room service
- Air conditioning
- Medical Facilities Service
- Shops and commercial services
- Safe deposit box
- Elevators
Other facilities
Guest rooms include the following facilities: Bathroom, Shower, Hairdryer, Direct dial telephone, TV, Minibar, Mini fridge, Carpeted floors, Safe, Balcony, Wi-fi, Toiletries, Bathrobes, Satellite TV, Cable TV, Number of bedrooms, NO Smoking rooms, NO Disability-friendly bathroom and NO Wheelchair-accessible.
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text:
Hi everybody, my name is Michiel and I am going to show you how to make an awesome looking laser engraver!
A couple of months ago, there was a CNC challenge here at instructables, while checking out the entries of that contest, I saw some pretty cool engraving machines and I thought: "Why shouldn't I make my own?". And so I did, but I didn't want to make someone else's project, I wanted to make my own. And so my story began... :)
This laser engraver uses a 1.8W 445nm laser module, of course, this is nothing compared to the industrial laser cutters who use lasers of (a lot) more than 50W. But this laser will do well for us. It can cut through paper and cardboard and it can engrave all kinds of wood. I haven't tested other materials yet, but I'm sure it can engrave many other materials. l will let you know! It has a large engraving surface of about 500x380mm.
Who can make this laser engraver? Everybody, if you are an engineer, a lawyer, a teacher or a student like me, you can build this thing! All you have to do is follow this instructable.
It took me about three months to design and build this engraving machine, including waiting for about a month for parts. I said three months, but, I'm only 16 years old, so I could only work in weekends.
++ As you may already know, this 'machine' uses a 1.8W laser with a wavelength of 445nm, the radiation of this laser is very dangerous for your eyes, even a reflection of it! So I strongly advise you to use safety goggles with the wavelength of the laser you use! ++