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Biomedical subjects

N S Fedarko

Publications and source records attributed to N S Fedarko.

At least 19 recordsLinked to original sources
PubMed2004

Serum levels of matrix extracellular phosphoglycoprotein (MEPE) in normal humans correlate with serum phosphorus, parathyroid hormone and bone mineral density.

Matrix extracellular phosphoglycoprotein (MEPE), a member of the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family, is primarily expressed in normal bone and has been proposed as a phosphaturic factor because of high expression and secretion in oncogenic hypophosphatemic osteomalacia tumors. In order to begin to address the role of MEPE in normal human physiology, we developed a competitive ELISA to measure serum levels of MEPE. The ELISA was used to characterize the distribution pattern in a population consisting of 114 normal adult subjects. The mean value of MEPE was 476 +/- 247 ng/ml and levels decreased significantly with increasing age. MEPE levels were also significantly correlated with serum phosphorus and parathyroid hormone (PTH). In addition, MEPE levels correlated significantly with measures of bone mineral density in the femoral neck and total hip in a subset of 50 elderly subjects. The results are consistent with MEPE being involved in phosphate and bone metabolism in a normal population.

Adult↗
PubMed2001

Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two members of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of genetically related proteins that are clustered on human chromosome 4. We present evidence that this entire family is the result of duplication and subsequent divergent evolution of a single ancient gene. The solution structures of these two post-translationally modified recombinant proteins were solved by one dimensional proton NMR and transverse relaxation times. The polypeptide backbones of both free BSP and OPN rapidly sample an ensemble of conformations consistent with them both being completely unstructured in solution. This flexibility appears to enable these relatively small glycoproteins to rapidly associate with a number of different binding partners including other proteins as well as the mineral phase of bones and teeth. These proteins often function by bridging two proteins of fixed structures into a biologically active complex.

Amino Acid Sequence↗
PubMed2001

Elevated serum bone sialoprotein and osteopontin in colon, breast, prostate, and lung cancer.

PURPOSE: Histological studies have shown that the two sialoproteins, bone sialoprotein (BSP) and osteopontin (OPN), are induced in multiple types of cancer. We have recently found that these proteins are bound in serum to complement factor H and that the complex must be disrupted to generate free protein to measure their total levels. We hypothesized that measuring total BSP and OPN levels would provide informative markers for the detection of cancer. EXPERIMENTAL DESIGN: As a proof of concept study, serum from patients with diagnosed breast, colon, lung, or prostate cancer (n = 20 for each type) as well as normal serum (n = 77) were analyzed using competitive ELISAs developed for BSP and OPN. Sensitivity, specificity, as well as positive and negative predictive values were determined for each sialoprotein and cancer type. The relationship between sensitivity and specificity was profiled by receiver operating characteristic curves. RESULTS AND CONCLUSIONS: Determined values for serum BSP in ng/ml were 285 +/- 19 for prostate, 373 +/- 19 for colon, 318 +/- 18 for breast, 155 +/- 11 for lung cancer sera, and 154 +/- 13 for normal sera. Values of OPN in ng/ml were 653 +/- 39 for prostate, 449 +/- 22 for colon, 814 +/- 53 for breast, 724 +/- 33 for lung, and 439 +/- 30 for normal sera. The assays provide a high degree of sensitivity and specificity that enables the detection of colon, breast, prostate, and lung cancer.

Biomarkers, Tumor↗
PubMed2000

Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack.

Metastatic cancer cells, like trophoblasts of the developing placenta, are invasive and must escape immune surveillance to survive. Complement has long been thought to play a significant role in the tumor surveillance mechanism. Bone sialoprotein (BSP) and osteopontin (OPN, ETA-1) are expressed by trophoblasts and are strongly up-regulated by many tumors. Indeed, BSP has been shown to be a positive indicator of the invasive potential of some tumors. In this report, we show that BSP and OPN form rapid and tight complexes with complement Factor H. Besides its key role in regulating complement-mediated cell lysis, Factor H also appears to play a role when "hijacked" by invading organisms in enabling cellular evasion of complement. We have investigated whether BSP and OPN may play a similar role in tumor cell complement evasion by testing to see whether these glycoproteins could promote tumor cell survival. Recombinant OPN and BSP can protect murine erythroleukemia cells from attack by human complement as well as human MCF-7 breast cancer cells and U-266 myeloma cells from attack by guinea pig complement. The mechanism of this gain of function by tumor cell expression of BSP or OPN has been defined using specific peptides and antibodies to block BSP and OPN protective activity. The expression of BSP and OPN in tumor cells provides a selective advantage for survival via initial binding to alpha(V)beta(3) integrin (both) or CD44 (OPN) on the cell surface, followed by sequestration of Factor H to the cell surface and inhibition of complement-mediated cell lysis.

Amino Acid Sequence↗
PubMed2000

Bone sialoprotein mediates human endothelial cell attachment and migration and promotes angiogenesis.

Bone sialoprotein (BSP) is a secreted glycoprotein primarily found in sites of biomineralization. Recently, we demonstrated that BSP is strongly upregulated in osteotropic cancers and particularly those that exhibit microcalcifications. BSP contains an Arg-Gly-Asp (RGD) motif found in other adhesive molecules that interact with cellular integrins. In bone, BSP has been shown to mediate the attachment of osteoblasts and osteoclasts via alpha(v)beta(3) integrin receptors. Ligands for alpha(v)beta(3) integrin are considered to play a central role during angiogenesis. Therefore, we used human umbilical vein endothelial cells (HUVECs) to study the potential role of BSP in angiogenesis. We found that purified eukaryotic recombinant human BSP (rhBSP) is able to promote both adhesion and chemotactic migration of HUVECs in a dose-dependent manner. These interactions involve HUVEC alpha(v)beta(3) integrin receptors and the RGD domain of BSP. Indeed, HUVECs attach to a recombinant BSP fragment containing the RGD domain, whereas this response is not observed with the same fragment in which RGD has been mutated to Lys-Ala-Glu (KAE). A cyclic RGD BSP peptide inhibits both adhesion and migration of HUVECs to rhBSP. Moreover, anti-alpha(v)beta(3) but not anti-alpha(v)beta(5) monoclonal antibodies also prevent BSP-mediated adhesion and migration of HUVECs. We observed that both rhBSP and the RGD BSP recombinant fragment stimulated ongoing angiogenesis on the chorioallantoic chick membrane assay. BSP angiogenic activity was inhibited by anti-alpha(v)beta(3) antibody, and the KAE BSP fragment was inactive. Our findings represent the first report implicating BSP in angiogenesis. BSP could play a critical role in angiogenesis associated with bone formation and with tumor growth and metastatic dissemination.

Cell Adhesion↗
PubMed2000

Interleukin-13 modulates collagen homeostasis in human skin and keloid fibroblasts.

Interleukin (IL)-13 has been implicated in the pathogenesis of various diseases characterized by fibrosis. We describe the effects of IL-13 on collagen homeostasis from normal (NF) and keloid (KF) fibroblasts and compare these effects with those of IL-4 and transforming growth factor (TGF)-beta(1). Total collagen generation was up-regulated in NF after 48 h of stimulation by IL-13; in KF, IL-13 stimulated a more rapid collagen response. The kinetics and magnitude of collagen generation induced by IL-13 were equivalent to those induced by similar concentrations of IL-4 and TGF-beta(1). Collagen type I production paralleled total collagen generation from both NF and KF; however, IL-4-induced collagen type I and total collagen production from KF was more transient than that induced by either IL-13 or TGF-beta(1). Procollagen 1alpha1 gene expression was induced in KF by stimulation with IL-13 for 24 h. Moreover, IL-13 was unique among these three cytokines in its ability to induce gene expression for procollagen 3alpha1. Finally, IL-13 inhibited IL-1beta-induced matrix metalloproteinase (MMP)-1 and MMP-3 production and enhanced tissue inhibitor of metalloproteinase (TIMP)-1 generation from NF; although similar effects were observed with IL-4, TGF-beta(1) transiently enhanced MMP-1 and MMP-3 generation without effecting TIMP-1. In KF, IL-13 and IL-4 inhibited MMP-3, whereas TGF-beta(1) enhanced MMP-3; TIMP-1 was unaffected by any of the three cytokines. These data demonstrate both the profibrotic effects of IL-13 on collagen homeostasis and the potential differential regulation of collagen homeostasis in fibroblast subtypes by IL-13.

Cells, Cultured↗
PubMed1998

Age-related changes in human oestrogen receptor alpha function and levels in osteoblasts.

Oestrogen receptors (ERs) are present in human osteoblasts and mediate anti-resorptive effects on bone. Human osteoblast-like cells derived from different aged healthy female donors not on hormone replacement therapy were utilized under well-defined conditions in vitro to investigate ER function and levels. Treatment with 0.1 nM oestradiol-17beta of cell strains derived from eight young women (less than 50 years of age) increased hydroxyproline levels significantly [an average (2.2+/-0.1 S.E.M.)-fold increase], whereas cells derived from nine older women (more than 50 years of age) were not significantly affected. Similarly, cell strains, derived from younger women, transfected with a consensus oestrogen-responsive element linked to chloramphenicol acetyltransferase exhibited a greater response to oestrogen than strains derived from older women. When basal ERalpha levels were measured by enzyme immunoassay and normalized on a per cell basis, osteoblast-like strains derived from younger women (n=24) had a mean value of 2.54+/-0.16 fmol of ERalpha per 10(6) cells. In contrast, strains derived from older women (n=20) had a mean value of 5.44+/-0.48 fmol of ERalpha per 10(6) cells. An age-related increase in ERalpha number was also observed in human skin-derived fibroblasts and directly in dermal biopsies from women not on hormone replacement therapy. The results demonstrate ligand concentration-dependent ERalpha induction and indicate a loss of receptor regulation and diminution of ligand-receptor signal transduction with increasing donor age.

Adolescent↗
PubMed1998

Receptor tyrosine kinase expression in human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are a heterogeneous population of cells derived from colony-forming units-fibroblastic (CFU-Fs). These cells reside in the bone marrow cavity and are capable of differentiating into several cell phenotypes including osteoblasts, chondroblasts, hematopoiesis-supporting stromal cells, and adipocytes. However, the factors that regulate the proliferation and differentiation of the BMSC population are for the most part unknown. Since many members of the receptor tyrosine kinase (RTK) family have been shown to participate in growth control of various mesenchymal cell populations, in this study we examined the expression and function of RTKs in the BMSC population. Degenerate oligonucleotides corresponding to two conserved catalytic domains of the RTK family and RT-PCR were used initially to determine which RTKs are expressed in the human BMSC (hBMSC) system. After subcloning the amplification product generated from mRNA of a multicolony-derived hBMSC strain, PDGF receptor (beta), EGF receptor, FGF receptor 1, and Axl were identified by DNA sequencing of 26 bacterial colonies. Furthermore, PDGF and EGF were found to enhance BMSC growth in a dose-dependent manner and to induce tyrosine phosphorylation of intracellular molecules, including the PDGF and EGF receptors themselves, demonstrating the functionality of these receptors. On the other hand, bFGF was found to have little effect on proliferation or tyrosine phosphorylation. Since single colony-derived hBMSC strains are known to vary from one colony to another in colony habit (growth rate and colony structure) and the ability to form bone in vivo, the expression levels of these RTKs were determined in 18 hBMSC clonal strains by semiquantitative RT-PCR and were found to vary from one clonal strain to another. While not absolutely predictive of the osteogenic capacity of individual clonal strains, on average, relatively high levels of PDGF-receptor were found in bone-forming strains, while on average, nonbone-forming strains had relatively high levels of EGF-receptor. Taken together, these results indicate that RTKs play a role in the control of hBMSC proliferation, and that the differential pattern of RTK expression may be useful in correlating the biochemical properties of individual clonal strains with their ability to produce bone in vivo.

Bone Marrow Cells↗
PubMed1998

Immortalization and characterization of bone marrow stromal fibroblasts from a patient with a loss of function mutation in the estrogen receptor-alpha gene.

A male patient with abnormal postpubertal bone elongation was shown earlier to have a mutation in both alleles of the estrogen receptor, resulting in a nonfunctional gene. Marrow stromal fibroblasts (MSFs) derived from this patient were called HERKOs (human estrogen receptor knock outs), and in order to obtain continuous HERKO cell lines, they were immortalized using a recombinant adenovirus-origin-minus SV40 virus. MSFs are unique cells because they support hematopoesis and contain a mixed population of precursor cells for bone, cartilage, and fat. Three established cell lines (HERKO2, HERKO4, and HERKO7) were characterized and compared with the heterogeneous population of nonimmortalized HERKOs for their osteogenic potential. We performed Northern analysis of matrix genes implicated in bone development and metabolism and an in vivo bone formation assay by transplanting the cells subcutaneously into immunodeficient mice. All three HERKO lines expressed high amounts of collagen 1A1, osteopontin, osteonectin, fibronectin, decorin, biglycan, and alkaline phosphatase. Except for osteopontin, expression of these genes was slightly lower compared with nonimmortalized HERKOs. In the in vivo bone formation assay, the heterogeneous population of nonimmortalized HERKOs formed bone with high efficiency, while the HERKO lines induced a high-density, bone-like matrix. Finally, all HERKO cell types secreted high levels of insulin-like growth factor I and interleukin-6 into the culture medium relative to cells of normal human subjects. In summary, these lines of HERKO cells retain several of the phenotypic traits of MSFs after immortalization, including matrix and cytokine production, and provide a valuable source of a unique human material for future studies involving estrogen action in bone and bone marrow metabolism.

Adult↗
PubMed1997

Age-related changes in effects of insulin-like growth factor I on human osteoblast-like cells.

The role of insulin-like growth factor I (IGF-I) in extracellular matrix metabolism was studied in both proliferating and confluent human osteoblast-like cultures derived from donors of different ages. In proliferating cultures, recombinant human (rh)IGF-I was found to increase the incorporation of [3H]thymidine in a dose- and age-dependent manner. To study cell proliferation dynamically, continuous growth curves with and without rhIGF-I were modelled by a modified logistic function. Increasing doses of rhIGF-I decreased the lag time and maximal growth rates, whereas plateau values decreased only at the highest dose (100 ng/ml). In post-proliferative cell strains, rhIGF-I (0.1-100 ng/ml) increased levels of type I collagen, biglycan and decorin, and to a smaller extent fibronectin and thrombospondin, whereas it decreased the levels of hyaluronan and a versican-like proteoglycan when protein and proteoglycan metabolism were followed by steady-state radiolabelling with [3H]proline, [3H]glucosamine or [35S]sulphate. These responses to rhIGF-I were found to be age-dependent, with osteoblast-like cells derived from younger patients being more responsive to rhIGF-I. When extracellular matrix turnover was analysed by pulse-chase experiments, rhIGF-I had no effect. The steady-state levels of collagen, decorin, hyaluronan and a versican-like proteoglycan for bone cells treated with rhIGF-I on day 7 in culture were equivalent to levels of these matrix components in untreated osteoblasts grown for 14 days. These results are consistent with rhIGF-I's altering cellular proliferative capacity and matrix synthesis, causing a change in the osteoblast differentiated state.

Aging↗
PubMed1997

2-Sulfotrehalose, a novel osmolyte in haloalkaliphilic archaea.

A novel 1-->1 alpha-linked glucose disaccharide with sulfate at C-2 of one of the glucose moieties, 1-(2-O-sulfo-alpha-D-glucopyranosyl)-alpha-D-glycopyranose, was found to be the major organic solute accumulated by a Natronococcus sp. and several Natronobacterium species. The concentration of this novel disaccharide, termed sulfotrehalose, increased with increasing concentrations of external NaCl, behavior consistent with its identity as an osmolyte. A variety of noncharged disaccharides (trehalose, sucrose, cellobiose, and maltose) were added to the growth medium to see if they could suppress synthesis and accumulation of sulfotrehalose. Sucrose was the most effective in suppressing biosynthesis and accumulation of sulfotrehalose, with levels as low as 0.1 mM being able to significantly replace the novel charged osmolyte. Other common osmolytes (glycine betaine, glutamate, and proline) were not accumulated or used for osmotic balance in place of the sulfotrehalose by the halophilic archaeons.

Disaccharides↗
PubMed1996

Long-term extracellular matrix metabolism by cultured human osteogenesis imperfecta osteoblasts.

Osteopenia due to deficient extracellular matrix synthesis is a hallmark of osteogenesis imperfecta (OI), Previous studies carried out within 72 h of osteoblast subculture, at an early stage of matrix synthesis, indicated that for osteoblasts derived from human OI patients the total amounts of collagen, osteonectin, and three proteoglycans were significantly reduced, while total amounts of thrombospondin, fibronectin, and matrix hyaluronan were elevated compared with age-matched controls. The current study was undertaken to follow OI osteoblast matrix metabolism as that matrix is synthesized, deposited, and matured. Steady-state metabolic radiolabeling was used to follow the metabolism of collagen, hyaluronan, and total proteoglycan by OI and normal osteoblasts for up to 5 weeks. Trabecular osteoblasts from non-OI controls showed an increase in total and matrix-associated collagen synthesis during the first and second week, reaching steady-state levels by week 4. In contrast, cultured OI osteoblasts did not increase either the total (medium + matrix-associated) or matrix-associated collagen during the entire 5-week period. Proteoglycan synthesis exhibited a pattern similar to that for collagen. OI-derived proteoglycans differed from controls in that levels in OI cultures did not reflect the normal time-dependent increase in total proteoglycan and proteoglycan matrix deposition. OI osteoblast hyaluronan synthesis was increased when compared with age-matched controls during 4 weeks of culture. In contrast, the ratios of calcium to phosphorus solublized from control and the OI extracellular matrix were not statistically different. Thus, with respect to the synthesis of collagen, proteoglycans, and hyaluronan, OI osteoblasts fail to parallel controls in depositing and elaborating extracellular matrix during 35 days in culture.

Adolescent↗
PubMed1995

Extracellular matrix stoichiometry in osteoblasts from patients with osteogenesis imperfecta.

In previous work, we compared the steady-state levels of specific matrix components in human bone cells derived from patients with osteogenesis imperfecta (OI) to those of age-matched controls. A remarkable finding was the observation that there was a reduction not only in the total levels of collagen, but also in osteonectin and three proteoglycans (a large chondroitin sulfate proteoglycan, biglycan, and decorin). This pattern was observed in patients with and without detectable collagen defects. More recent analysis of extracellular matrix composition have yielded that, compared with age-matched controls, bone cells from OI patients produced higher steady-state levels of fibronectin and thrombospondin. The percentage of these two proteins incorporated into the cell layer pool was also higher in OI than in age-matched controls. In addition, the steady-state levels of hyaluronan and a heparan sulfate proteoglycan were analyzed in both OI and age-matched controls. Although the total (medium + cell layer) steady-state levels of hyaluronan were reduced by 1/3, the percentage of the hyaluronan in the cell layer pool of patients with OI increased between 100-250% of age-matched control. Thus the matrix elaborated by human OI bone cells is not only quantitatively different but also qualitatively distinct from that of age-matched controls. Not only have specific bone cell matrix components (collagen, osteonectin, the large chondroitin sulfate proteoglycan, biglycan, and decorin) been found to be present in reduced levels in OI bone cells, but some matrix components (thrombospondin, fibronectin, and hyaluronan) have also been found to be present in elevated levels in the matrix of OI cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Cell proliferation of human fibroblasts and osteoblasts in osteogenesis imperfecta: influence of age.

Clinical studies indicate that as a group, osteogenesis imperfecta (OI) subjects are shorter than age- and sex-matched controls. Not only somatic growth, but also cellular growth appears to be impaired, and these may be related to defects in extracellular matrix common to this disorder. We have investigated the growth characteristics of dermal fibroblasts and trabecular osteoblasts isolated from patients with OI and control subjects of various ages. Cell growth curves and cell doubling times were determined by measuring cell number using crystal violet dye binding. Growth curves were modeled by a modified logistic function, the three parameters of which are markers for biologically relevant growth parameters: the plateau value or upper asymptote, which reflects the maximum cell density upon confluence; the maximal growth rate (microM); and the lag time. Both normal human fibroblasts and osteoblasts showed an age-dependent decrease in microM. Normal fibroblasts exhibited no age-dependence to their upper asymptote or lag time. Fibroblasts derived from patients with OI did not have significantly different upper asymptote values microM, or lag times when compared with normal fibroblasts. Normal osteoblasts had a decrease in upper asymptote, decrease in microM, but a relatively constant lag time with increasing age. In contrast, OI osteoblast microM was decreased relative to that of normal subjects. For osteoblasts from OI patients, decreased microM appeared unrelated to the age of the subject, whereas OI fibroblasts did exhibit an age-dependent decrease in microM. The percentage of collagenase-digestible protein (a measure of collagen synthesis) produced by normal human fibroblasts correlated well with microM. Treating normal human osteoblasts with the proline analogue 3,4-dehydroproline, which destabilizes collagen triple helix formation and alters collagen synthesis, secretion, and turnover, also decreased microM. A dose response to varying concentrations of 3,4-dehydroproline was observed for normal human bone cell microM. These data suggest a link between type I collagen synthesis and cellular proliferation.

Aging↗
PubMed1995

The X-chromosomal human biglycan gene BGN is subject to X inactivation but is transcribed like an X-Y homologous gene.

We report the mRNA and protein expression levels of human biglycan (BGN) in patients with different numbers of sex chromosomes. BGN maps to the distal long arm of the X chromosome, band Xq28, near the second pseudoautosomal region. BGN expression levels are reduced in 45,X Turner patients and increased in patients with additional sex chromosomes. This is suggestive of a pseudoautosomal gene or a gene that escapes X inactivation and that has an active Y chromosomal copy. However, we also provide evidence from hybrid cell lines that BGN is subject to X inactivation and that there is no homolog on the Y chromosome. This evidence excludes an escape from X inactivation. Moreover, additional Y chromosomes increase BGN expression levels, despite the absence of a Y chromosomal BGN gene. Therefore, another explanation has to be invoked. The "pseudoautosomal expression" of BGN may be attributed to a gene or genes that escape X inactivation and that regulate the transcriptional activity of BGN. This is the first report concerning an X chromosomal gene that does not show the conventional correlation between gene dosage and expression rate known from other X chromosomal genes.

Adult↗
PubMed1995

OIM and related animal models of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is characterized by fragile bones, skeletal deformity, and growth retardation. This heritable disorder of connective tissue is the result of mutations affecting the COL1A1 and COL1A2 genes of type I collagen. Progress in OI research has been limited because of dependence on human fibroblast and osteoblast specimens and the absence of a naturally occurring animal model for this genetic disorder. Recent technology in molecular biology has led to the development of transgenic models of OI based on site directed mutagenesis of type I collagen genes. OIM is a naturally occurring model which incorporates both the phenotypic and biochemical defects of moderate to severe osteogenesis imperfecta. This powerful tool permits the development of models based on different type I collagen mutations. The collagen type I mutation in OIM is a C propeptide deletion which impairs the production of normal pro-alpha2(I). Tissues in OIM contain only [pro-alpha1(I)]3 homotrimer. Thus, although several animal models are now available for research in osteogenesis imperfecta few are viable or fully mimic human disease disorders. OIM duplicates the phenotype and biochemistry of human disease and has a normal life span.

Animals↗