Biomedical subjects

Karen E Yates

Publications and source records attributed to Karen E Yates.

14 recordsLinked to original sources

PubMed2006

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling.

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.

Adult↗

PubMed2006

Identification of cis and trans-acting transcriptional regulators in chondroinduced fibroblasts from the pre-phenotypic gene expression profile.

Cell differentiation is regulated via expression of successive sets of genes. In an in vitro model of chondrocyte differentiation, human dermal fibroblasts (hDFs) cultured in collagen sponges are induced to express cartilage matrix genes after 7 days' culture with demineralized bone powder (DBP). A shift in expression of many other genes occurs within 3 days, before chondroblast phenotypic genes are detectable. In this study, the pre-chondrogenic gene expression profile was used as a starting point to derive information on transcriptional regulation of chondrocyte differentiation induced by DBP. Putative cis regulatory elements were identified by comparing promoter regions from three genes that are highly upregulated in chondroinduced hDFs (BIGH3, COL1A2, and FN1) [Zhou, S., Glowacki, J., Yates, K.E, 2004. Comparison of TGF-beta/BMP pathways signaled by demineralized bone powder and BMP-2 in human dermal fibroblasts. J. Bone Min. Res. 19, 1732-1741] and whose products are known to interact in the matrix [Kim, J.E., et al., 2002. Molecular properties of wild-type and mutant betaIG-H3 proteins. Investig. Ophthalmol. Vis. Sci. 43, 656-661]. The effect of DBP on nuclear protein binding to cis elements was measured with an array-based assay. Nuclear extracts from hDFs cultured in DBP/collagen sponges for 3 days showed increased binding to several cis elements belonging to the families that were identified by promoter analysis. Of note, those elements represented targets of both signal-activated and developmentally regulated transcription factors. Direct measurement of mRNAs showed increased gene expression of both types of transcription factors in chondroinduced hDFs, including NFKB2 (290% of control), RELA (160%), and GATA2 (190%). Moreover, DBP increased gene expression of chondrogenic transcription factors SOX9 (160% of control) and RUNX2 (180%). Immunoblot analysis showed that DBP increased both expression (200% of control) and phosphorylation (300%) of the Creb protein, a transcription factor that is downstream of several signal transduction pathways. Inhibition of protein kinase A, protein kinase C, or MAP kinase in hDFs cultured in DBP/collagen sponges reduced induction of BIGH3 to approximately 50% of control. These results suggest that both signal-activated and developmentally controlled transcriptional mechanisms contribute to chondroinduction of hDFs by DBP.

Binding Sites↗

PubMed2005

Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges.

Demineralized bone implants have been used for many types of craniomaxillofacial, orthopedic, periodontal, and hand reconstruction procedures. In previous studies, we showed that demineralized bone powder (DBP) induces chondrogenesis of human dermal fibroblasts in a DBP/collagen sponge system that optimized interactions between particles of DBP and target cells in cell culture. In this study, we test the hypothesis that DBP promotes chondrogenesis or osteogenesis of human marrow stromal cells (hMSCs) in 3-D collagen sponge culture, depending upon the culture conditions. We first confirmed that hMSCs have chondrogenic potential when treated with TGF-beta, either in 2-D monolayer cultures or in 3-D porous collagen sponges. Second, we found that DBP markedly enhanced chondrogenesis in hMSCs in 3-D sponges, as assessed by metachromasia and expression of chondrocyte-specific genes AGGRECAN, COL II, and COL X. Human dermal fibroblasts (hDFs) were used to define mechanisms of chondroinduction because unlike hMSCs they have no inherent chondrogenic potential. In situ hybridization revealed that hDFs vicinal to DBPs express chondrocyte-specific genes AGGRECAN or COL II. Macroarray analysis showed that DBP activates TGF-beta/BMP signaling pathway genes in hDFs. Finally, DBP induced hMSCs to express the osteoblast phenotype when cultured with osteogenic supplements. These studies show how culture conditions can influence the differentiation pathway that human marrow stromal cells follow when stimulated by DBP. These results support the potential to engineer cartilage or bone in vitro by using human bone marrow stromal cells and DBP/collagen scaffolds.

Aggrecans↗

PubMed2005

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes.

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS(+3)) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.

Aggrecans↗

PubMed2005

Spatial and temporal changes in the subcellular localization of the nuclear protein-tyrosine kinase, c-Fes.

Tyrosine phosphorylation has emerged as a mechanism to control cellular events in the nucleus. The c-Fes protein-tyrosine kinase is an important regulator of cell growth and differentiation in several cell types, and is found in the nucleus of hematopoietic cells. In this study, we showed nuclear localization of c-Fes in both hematopoietic (K562, TF-1, HEL, U937, and HL-60) and nonhematopoietic cell lines (293T, CaOv3, TfxH, MG-63, HeLa, DU-145) by immunofluorescence and confocal microscopy. c-Fes showed striking changes in subcellular localization at specific stages of mitosis. In interphase cells, the intranuclear distribution of c-Fes was diffuse with occasional bright foci. Some c-Fes was present in the cytosol after breakdown of the nuclear membrane, in prometaphase. At prometaphase and metaphase c-Fes was also associated with the chromosomes, in a punctate pattern that partially overlapped with the centromere. Further comparison with proteins that are known components of the kinetochore suggested that some c-Fes protein was located at the centromeric alpha-satellite DNA, between the kinetochores. At anaphase and telophase, c-Fes was entirely cytoplasmic and no protein was found associated with the chromosomes. The timing of c-Fes' appearance at the centromere coincides with the period of kinetochore assembly. These data suggest that c-Fes is recruited to the kinetochore during mitosis.

Animals↗

PubMed2005

Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

Animals↗

PubMed2005

Wnt influence on chondrocyte differentiation and cartilage function.

The Wnt signaling network regulates chondrocyte differentiation, proliferation, and maturation during embryonic limb development. In this review, we summarize studies of Wnt signaling during the chondrocyte life cycle in avian and mammalian systems, both before and after birth. Recent reports that implicate abnormal Wnt signaling as a contributing factor to pathogenic joint conditions are also discussed. In addition, we show new data that suggests Wnt signaling is active in adult cartilage. Overall, it appears that the Wnt network has dual roles in cartilage, as has been described in other tissues: it is an important regulator of chondrocyte development, but deregulated signaling is detrimental to mature tissues and may lead to disease.

Animals↗

PubMed2004

Comparison of TGF-beta/BMP pathways signaled by demineralized bone powder and BMP-2 in human dermal fibroblasts.

UNLABELLED: Demineralized bone induces chondrogenic differentiation of human dermal fibroblasts in vitro. Analyses of signaling gene expression showed that DBP and BMP-2 regulate common and distinct pathways. Although BMP-2 was originally isolated as a putative active factor in DBP, rhBMP-2 and DBP do not affect all the same genes or in the same ways. INTRODUCTION: Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in 3D culture, but the initiating mechanisms have not been identified. We tested the hypotheses that DBP would affect expression of signaling genes and that DBP's effects would differ from the effects of bone morphogenetic proteins (BMPs). MATERIALS AND METHODS: A chondroinduction model was used in which hDFs were cultured with and without DBP in a porous collagen sponge. BMP-2 was delivered in a square of absorbable collagen felt inserted into a collagen sponge. Total RNA was isolated after 3 days of culture, a time that precedes expression of the chondrocyte phenotype. Gene expression was evaluated with two targeted macroarray screens. Effects of DBP and rhBMP-2 were compared by macroarray, RT-PCR, and Northern hybridization analysis of selected genes in the transforming growth factor (TGF)-beta/BMP signaling pathways. RESULTS: By macroarray analysis of 16 signal transduction pathways, the following pathways were modulated in hDFs by DBP: TGF-beta, insulin/LDL, hedgehog, PI3 kinase/AKT, NF-kappaB, androgen, retinoic acid, and NFAT. There was convergence and divergence in DBP and rhBMP-2 regulation of genes in the TGF-beta/BMP signaling pathway. Smad target genes were the predominant group of DBP- or rhBMP-2-regulated genes. Several genes (IGF-BP3, ID2, and ID3) showed similar responses (increased expression) to DBP and rhBMP-2. In contrast, many of the genes that were greatly upregulated by DBP (TGFBI/betaig-h3, Col3A1, TIMP1, p21/Waf1/Cip1) were barely affected by rhBMP-2. CONCLUSION: These findings indicate that multiple signaling pathways are regulated in fibroblasts by DBP, that one of the major pathways involves Smad target genes, and that DBP and rhBMP-2 elicit different gene expression responses in hDFs. Although BMP-2 was originally isolated as a putative inductive factor in DBP, rhBMP-2 and DBP do not affect all the same genes or in the same ways.

Animals↗

PubMed2004

Demineralized bone alters expression of Wnt network components during chondroinduction of post-natal fibroblasts.

OBJECTIVE: The Wnt family of secreted proteins, their receptors (Fzd proteins) and antagonists (secreted Fzd-related proteins, or Sfrp) regulate chondrocyte differentiation and chrondrogenesis during embryonic development. Here, the hypothesis that the Wnt regulatory network contributes to chondrocyte differentiation of post-natal cells was tested in an in vitro model of chondroinduction by demineralized bone powder (DBP). DESIGN: Human dermal fibroblasts (hDFs) were cultured in porous, three-dimensional (3D) collagen sponges with or without chondroinductive DBP. In some experiments, lithium chloride (LiCl), an agonist of the Wnt/beta-catenin signaling pathway, was added to the culture media. Sponges were cultured for intervals (0.5-21 days) before processing for molecular, histologic, and biochemical analyses. Expression of wnt, fzd, and sfrp genes was characterized by semi-quantitative RT-PCR. Fibroblasts' contacts with DBP were documented by histology. Accumulation of proteoglycan in extracellular matrix was evaluated by histology (metachromasia in toluidine blue-stained sections) and quantitative immunoassay (chondroitin 4-sulfate ELISA). RESULTS: Expression of 15 wnt, fzd, and sfrp family members was detected in hDFs by RT-PCR. A subset of those genes (wnt2b, wnt5b, wnt10b, fzd6, fzd7) showed altered expression in hDFs exposed to DBP for 3 days. wnt and fzd gene expression was not altered before hDFs contacted the DBP within the collagen sponge. Human DFs cultured in plain collagen sponges and treated with LiCl accumulated significantly more metachromatic matrix than NaCl-treated controls on day 10, and showed a trend towards increased matrix chondroitin-4 sulfate content. CONCLUSIONS: These data suggest that changes in Wnt signaling contribute to chondroinduction of post-natal fibroblasts by DBP. This is the first evidence that Wnt components, which are essential regulators of pre-natal chondrocyte differentiation, may also influence post-natal chondrocyte differentiation induced by DBP.

Bone Demineralization Technique↗

PubMed2004

Inferred functions of "novel" genes identified in fibroblasts chondroinduced by demineralized bone.

Little is known about the cellular mechanisms that control postnatal chondrocyte differentiation. As a first step towards identifying those mechanisms, gene expression shifts were characterized in an in vitro model of chondroinduction. In previous studies, several functional classes of genes (cytoskeletal and matrix elements, cell adhesion proteins, peptide growth factors, and signal transduction proteins) were found to be altered in human dermal fibroblasts (hDFs) cultured in porous collagen sponges with chondroinductive demineralized bone powder (DBP) for 3 days. In addition, a number of "novel" sequences were identified. In this study, molecular techniques were combined with computational methods to characterize those sequences. Gene expression of all 10 novel sequences tested was found in hDFs by RT-PCR. The sequences were compared to the human genome, and their cellular functions were inferred from genes that mapped to the same chromosomal coordinates. Only one of the novel sequences contained a protein-coding region (kinesin superfamily protein 26B). The others contained 3' untranslated (osteonectin, alpha-V integrin, RAP2B) or other untranslated regions (PTPN21, GAS6) of mRNAs. The cellular functions of the DBP-regulated genes described in this study fall into similar categories as those previously identified. These results provide new details on the cellular response of hDFs exposed to DBP.

3' Untranslated Regions↗

PubMed2004

New chondrocyte genes discovered by representational difference analysis of chondroinduced human fibroblasts.

This report includes a review of the potential for gene expression analyses to provide new information for solving problems in skeletal repair and regeneration. It focuses on two approaches: high-throughput gene array methods and representational difference analysis (RDA). The principles underlying these methods are presented with experimental tutorials and some applications. Second, this report includes a review of results from applying both approaches to an in vitro model of postnatal chondroinduction by demineralized bone powder (DBP). Human dermal fibroblasts (hDFs) cultured with DBP acquire a chondroblast phenotype and express cartilage-specific matrix proteins after 7 days. We used cDNA macroarrays and RDA to identify the genes that were altered prior to expression of the chondroblast phenotype, i.e., after only 3 days' culture with DBP. Using a strategy of data management and reduction based upon biological functions, we reported several functional families of genes (cytoskeletal elements, protein synthesis/trafficking, and matrix molecules and their modifiers) that are upregulated during chondroinduction of hDFs. Together with histological and biochemical evidence of the chondroblast phenotype, the gene expression patterns indicate that there are specific stages of induced chondrocyte differentiation in this experimental system. Third, this report includes a new study, in which DBP-regulated genes were used as a data base to derive new information on the cell biology of chondrocytes. The objective was to determine whether a set of genes expressed during induction of chondrocyte differentiation is also expressed by mature articular chondrocytes. Our search of the literature for 59 of the DBP-regulated genes disclosed that expression of 20 of them (33%) had been documented in mature cartilage or chondrocytes. Of the 39 genes not previously documented in cartilage, 11 were tested by RT-PCR and all were found to be expressed in freshly isolated adult human chondrocytes. This review and these new data show how the strategy of high-throughput methods and functional data reduction can expand our knowledge of chondrocyte cell biology.

Animals↗

PubMed2003

Altered expression of connective tissue genes in postnatal chondroinduced human dermal fibroblasts.

In a novel model for postnatal chondroinduction, normal human dermal fibroblasts (hDFs) cultured with demineralized bone powder (DBP) express chondrocyte features after 7 days. Representational difference analysis (RDA) prior to overt chondroblastogenesis (3 days) revealed altered expression of connective tissue genes (collagens, collagen receptors, and post-translational enzymes). Northern or RT-PCR analysis at 3, 7, 14, or 21 d showed different expression patterns for those genes. COL3A1 was transiently upregulated, whereas upregulation of COL11A1, integrin alpha-11, lysyl oxidase, and lysyl hydroxylase 2 mRNAs persisted for 7 days. Downregulation of decorin was sustained for 21 d. The expression of the post-translational enzymes induced by DBP was unique when compared with human skin and human articular chondrocytes. Thus, the data suggest an "induced chondroblast" stage with a unique connective tissue gene expression profile that may result in a matrix supportive of chondrogenesis by postnatal cells.

Aged↗

PubMed2002

Nerve growth factor is expressed in rat femoral vein.

PURPOSE: Entubulization is a well known method of nerve repair for defects too large to be reconstructed by direct suturing without tension. Vein grafts and alloplastic tubes have been used for entubulization in peripheral and cranial nerves, but the mechanism by which they promote healing is poorly understood. The overall hypothesis of this laboratory is that nerve growth factor (NGF) plays an important role in nerve regeneration after entubulization with a vein graft. The purpose of this pilot study was to localize NGF protein expression in the rat femoral vein. MATERIALS AND METHODS: Sciatic nerves and femoral veins were harvested from adult male Sprague-Dawley rats. Femoral arteries were also collected and used for comparison and validation of the analysis. All specimens were fixed in paraformaldehyde and embedded in paraffin. Specimens were either stained with hematoxylin and eosin or used for immunohistochemical reaction with anti-NGF antibody. RESULTS: Sciatic nerve was used as a positive control to identify the monofascicular architecture with hematoxylin and eosin and to document the positive immunohistochemical reaction. NGF immunoreactivity was present in the tunica intima and tunica adventitia of femoral vein and artery but not in the tunica media. CONCLUSION: The results of this pilot study indicate that NGF is detectable in both the intimal and adventitial layers of the rat femoral vein and artery but not in the smooth muscle wall. These findings suggest that vein grafts could potentially promote nerve regeneration by supplying NGF to the injured nerve.

Animals↗

PubMedDate not supplied

Gene expression changes in an in vitro model of chondroinduction: a comparison of two methods.

There are many useful technologies to describe patterns of gene expression that occur during tissue repair and regeneration. Results from different methods used in one experimental setting are not often compared. In this case study of chondrogenesis, we compare two methods to identify differentially expressed genes, representational difference analysis and targeted macroarray analysis, as a model for investigating genes that may be relevant to tissue repair. We sought to identify genes whose expression was altered when human dermal fibroblasts were cultured in a three-dimensional, porous collagen sponge with the chondroinductive agent, demineralized bone. Both representational difference analysis and macroarray experiments revealed several functional families of genes as up-regulated or down-regulated in chondroinduced fibroblasts. An advantage of representational difference analysis is that altered expression of specific mRNA transcripts can be revealed. In this example, representational difference analysis uncovered the up-regulation of a specific transcript of Wnt5a in fibroblasts cultured with demineralized bone. Representational difference analysis is limited, however, as there can be false negatives for genes not readily amplified by polymerase chain reaction. We conclude that small arrays containing functional classes of genes can be used to ask specific, hypothesis-driven questions at minimal cost. It may be prudent, however, to use more than one method to survey differences in gene expression in order to validate and expand findings.

Bone and Bones↗