Germline mutations in genes encoding members of the transforming growth factor-β (TGF-β)/<em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) superfamily are causal for two hereditary vascular disorders, hereditary hemorrhagic telangiectasia (HHT) and heritable pulmonary arterial hypertension (PAH). When the two diseases coexist, activin A receptor type II-like kinase-1 (ACVRL1) gene mutations are usually identified. We report a remarkable ACVRL1 germinal and somatic mosaicism characterized by the presence of two distinct mutant alleles and a non-mutant ACVRL1 allele in a woman diagnosed with PAH at the age 40. She also met the Curaçao diagnostic criteria for HHT based on additional findings of telangiectases, epistaxis and arteriovenous malformations. Mutation analysis of ACVRL1 identified two adjacent heterozygous deleterious mutations within exon <em>10</em>: c.1388del (p.Gly463fsX2) and c.1390del (p.Leu464X) in a region enriched by mutation-associated DNA motifs. The mother transmitted the c.1388del to one child and the c.1390del to two children confirming germinal mosaicism. Allele-specific polymerase chain reaction analysis showed that c.1388del is the predominant mutation in lymphocytes of the index case. Haplotype analysis revealed that both mutant alleles have a common chromosomal origin which is distinct from that of the mother's non-mutant ACVRL1 allele. These distinct mutant alleles in tissues and germline could have arisen by DNA structure-mediated events occurring in the early stages of the mother's embryogenesis, prior to the segregation of her germline, which ultimately led to the independent transmission of each allele. These highlight the complexity of genomic events occurring during early embryogenesis and the consequences of mutational mosaicism upon pathogenic variability.

The aim of this study was to evaluate and compare the effects of two commercial mineral trioxide aggregate (MTA) cements (ProRoot MTA and MTA Angelus) on transforming growth factor (TGF)-beta1 and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2 levels produced by cultured human gingival fibroblasts (HGFs). Human gingival tissues were obtained from individuals with healthy periodontium. HGFs were grown at 37 degrees C in humidified atmosphere of 5% CO(2) in Dulbecco's modified Eagle's medium, supplemented with <em>10</em>% fetal calf serum, penicillin, and streptomycin. After 24 and 72 hours of exposure to the MTA products, HGF viability was determined by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay. TGF-beta1 and BMP-2 levels in cell-free culture media were determined by enzyme-linked immunosorbent assay. Cell viability of the test groups was significantly lower than that of control at 24 and 72 hours (p < 0.05) but showed an increase at 72 hours (p < 0.05). Both test groups showed increased TGF beta-1 levels at 72 hours (p < 0.05), whereas the MTA Angelus group displayed higher TGF beta-1 levels than control and ProRoot MTA groups at 24 and 72 hours (p < 0.05). At 24 hours, BMP-2 levels of the ProRoot group were significantly higher than that of MTA Angelus (p < 0.05). Both test materials increased the BMP-2 levels within time (p < 0.05) and displayed similar levels at 72 hours (p>> 0.05). These results suggest that both MTA products are capable of stimulating HGF to produce BMP-2, whereas the stimulatory effect for TGF beta-1 is material dependent.

The oocyte derived growth differentiation factor (GDF) 9 and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 15 (BMP15; also known as GDF9b) are essential for normal follicular growth. However, little is known about expression of these factors during ovarian development. Therefore, we determined the ontogeny of expression of GDF9 and BMP15 mRNA in the developing ovary of the brushtail possum. Ovaries were collected from pouch young (n=3-5 per group) around times of key developmental events namely: (1) morphological sexual differentiation (i.e. days 1-5 following birth), (2) after sexual differentiation (i.e. days <em>10</em>-15), (3) before and during initiation of germ-cell meiosis (i.e. days 22-45), (4) shortly after initiation of follicular growth (i.e. days 78-85), (5) during preantral follicular growth (i.e. days 96-113) and (6) during antral follicular growth (i.e. days 155-190). Ovaries were also collected from three juvenile and four adult animals and gene expression was determined by in situ hybridization. The mRNAs encoding GDF9 and BMP15 were first observed in oocytes of newly-formed primordial follicles (i.e. days 78-85). Expression of both mRNAs was restricted to the oocyte and was present in follicles irrespective of whether they were non-growing primordial follicles or undergoing preantral or antral development. Thus, since the mRNAs encoding GDF9 and BMP15 were not observed until follicular formation, it is unlikely that these <em>proteins</em> have any role in early germ cell development. Nevertheless, the findings that the mRNAs encoding both <em>proteins</em> were observed in oocytes from the primordial stage of follicular formation suggest a possible role for these <em>proteins</em> in the maintenance of primordial follicles as well as a key role during follicular development. These results highlight important species differences in the ontogeny of expression of GDF9 and BMP15 between possums and other species such as the human, sheep or rat.

Although the <em>bone</em> <em>morphogenetic</em> <em>proteins</em> stimulate chondrogenesis, little is known regarding their expression and regulation in growth-plate chondrocytes. The expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 was examined in chick growth-plate chondrocyte cultures. Low basal levels of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 mRNA and <em>protein</em> expression were stimulated by increasing doses of all-trans retinoic acid, a metabolite of vitamin A. The addition of <em>10</em> microM retinoic acid resulted in approximately a 6-fold increase in <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 mRNA levels. In contrast, other growth regulators, including basic fibroblast growth factor, transforming growth factor-beta, vitamin D, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-6, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7, and parathyroid hormone-related peptide, did not alter <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 transcript levels. The increase in <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 transcripts, although present at 6 hours, was maximal following a 12-hour exposure to retinoic acid. Retinoic acid induction of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 transcript levels was dependent on <em>protein</em> synthesis because the induction could be blocked by cyclohexamide. In maturationally distinct subpopulations of chondrocytes separated by countercurrent centrifugal elutriation, retinoic acid markedly induced <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 mRNA levels in the least differentiated chondrocytes but had no effect in the most terminally differentiated hypertrophic chondrocytes. Immunohistochemical localization of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 demonstrates its expression throughout the developing and adolescent growth plate consistent with the constitutive pattern of expression seen in isolated chondrocytes. The addition of exogenous <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 to chondrocyte cultures stimulated maturation in undifferentiated chondrocyte populations. The data support a role for <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 as an autocrine regulator of chondrocyte maturation in the growth plate. Regulation of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 by retinoic acid may be important in normal growth and development as well as in pathologic conditions of an excess or deficiency of vitamin A.

Implantation of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) using a carrier or by BMP gene transfer into rodent muscle can induce <em>bone</em> formation. Implanted BMP becomes bioactive immediately after implantation. In BMP gene transfer, there is a time-lag between the secretion of gene products and <em>bone</em> formation. We analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ectopic <em>bone</em> formation by using semi-quantitative RT-PCR. A plasmid vector containing mouse BMP4 gene (pCAGGS-BMP4) was transferred into the gastrocnemius muscles of mice using electroporation. Histological examination revealed the process of endochondral <em>bone</em> formation in the pCAGGS-BMP4 transferred muscles. As chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen X the gene maximal expression was on day <em>10</em>. As osteogenic markers, osteocalcin (OCN), <em>bone</em> sialo<em>protein</em> (BSP) and osteopontin (OPN) gene expressions were clearly detected from day <em>10</em> and then increased by day 14. In conclusion, the present study proved that ectopic <em>bone</em> formation by BMP4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized <em>bone</em> matrix.

Insulin-like growth factors (IGFs) are found in human circulation predominantly as part of a growth hormone (GH)-dependent complex of 125-150 kD, which is composed of three subunits: IGF-I or IGF-II, an acid stable IGF binding <em>protein</em> (IGFBP)-3, and an acid labile subunit (ALS). Although recent studies demonstrate that a number of cell types in culture secrete IGFs and IGFBP-3, very little is known with regard to the origin of circulating ALS. To test the hypothesis that human <em>bone</em> cells (HBCs), which produce abundant amounts of IGF-II and IGFBP-3, also produce ALS, we measured the IGF-I, IGF-II, IGFBP-3, and ALS levels using specific radioimmunoassays (RIAs) in the conditioned medium (CM) of untransformed normal HBCs and SaOS-2 osteosarcoma cells treated with various effectors (IGF-II, osteogenic <em>protein</em>-1 [OP-1, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7] and human GH) for 48 h. No detectable levels (< 3 ng/ml) of ALS were found in the CM of various HBC types under basal conditions. In contrast, CM collected from liver explants in culture contained significant amount of ALS prepared and assayed under identical conditions. The IGF-I level was also undetectable in the CM of various HBC types. In the IGF-II (3, 30 ng/ml)-treated HBC CM, the IGFBP-3 level was increased in a dose-dependent manner but neither IGF-I nor ALS could be detected. In the SaOS-2 cell culture, OP-1 (1, <em>10</em>0 ng/ml) increased both IGF-II and IGFBP-3 secretion but neither ALS nor IGF-I secretion. Treatment of HBCs with GH (1, <em>10</em>, <em>10</em>0 ng/ml) had no significant effect on the secretion of either IGF-I, IGF-II, IGFBP-3, or ALS.(ABSTRACT TRUNCATED AT 250 WORDS)

The novel mammalian gene Crim1 encodes a transmembrane bound <em>protein</em> with similarity to the secreted <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) antagonists, vertebrate Chordin, and its Drosophila homologue short gastrulation. Crim1 is expressed in the neural tube in mouse in a restricted pattern, but its function in central nervous system development is largely unknown. We isolated the chicken Crim1 orthologue and analyzed its expression in the developing neural tube. Chicken CRIM1 shares strong homology to human/mouse CRIM1 and C. elegans CRIM1-like <em>proteins</em>. Crim1 is expressed in a similar but not identical pattern to that in the developing spinal cord of mouse, including the notochord, floor plate, motor neurons, and the roof plate. Unlike follistatin, a secreted inhibitor of BMPs, in ovo electroporation of CRIM1, as a full-length transmembrane bound or secreted ectodomain was not sufficient to disrupt early patterning of the neural tube. However, ectodomain CRIM1 overexpression leads to an approximate 50% decrease in populations of specific ventral neuronal populations, including ISL-1(+) motor neurons, CHX-<em>10</em>(+) V1, and EN-1(+) V2 interneurons.

The aims of the present study were to fabricate a novel porous polylactic acid (PLLA) composite scaffold and evaluate the capacity of the scaffold in carrying recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (rhBMP2) for engineering <em>bone</em> formation. The structures of the PLLA scaffolds were evaluated by SEM and the controlled release of rhBMP2 from the composite scaffolds was assayed by ELISA. <em>Bone</em> induction by the scaffolds loaded with or without rhBMP2 was performed in the calf muscle of twenty Wistar rats for 3, 7, <em>10</em>, 14, and 28 days. Tissue specimens were examined by Masson's trichrome and von Kossa stainings, and immunohistochemistry of <em>bone</em> <em>proteins</em>. Our results indicated that a moderate foreign body reaction was found in control scaffolds, which lasted for 4 weeks. The addition of rhBMP2 to this novel scaffold dramatically alleviated the adverse responses to PLLA. Enhanced deposition of collagen matrix and endochondral formation were observed in rhBMP2-PLLA scaffolds at 7-<em>10</em> days, compatible with an early release of rhBMP2 in the composite scaffolds. <em>Bone</em> sialo<em>protein</em> and osteopontin were demonstrated simultaneously. Von Kossa staining was observed in the test group at <em>10</em>-14 days. In conclusion, the PLLA scaffolds exhibited the capability of carrying rhBMP2 for inducing <em>bone</em> formation within 2 weeks. These results suggest that rhBMP2-PLLA scaffold may be applicable in tissue engineering.

The purpose of this study was to evaluate a hydroxyapatite (HA)-collagen (Col)-<em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) composite as an osteoinductive <em>bone</em> substitute. Partially-purified BMP from bovine <em>bone</em> was mechanically mixed with highly purified type I collagen from calf dermis and then subsequently mixed with pure synthetic hydroxyapatite granules or block. The HA-Col-BMP composite, or the HA-Col composite as a control, was implanted in a surgically-induced mandible <em>bone</em> defect (6 x 7 x <em>10</em> mm) in an adult Japanese monkey. The mandible was excised three months after implantation and studied histologically. The BMP-containing implant induced much more new <em>bone</em> than the control implant in all experimental animals of each group, with either HA granules or HA block. Newly formed <em>bone</em> was attached tightly to HA and infiltrated deeply into the pores of the HA of the BMP-containing implant, while fibrous tissue existed between the host <em>bone</em> and HA in the control implant. Thus, we conclude that an HA-Col-BMP composite could be a superior biomaterial for a <em>bone</em> substitute.

In adult rabbits, mid-diaphyseal segments of the radius or ulna were excised to produce defects greater than the critical size for spontaneous <em>bone</em> repair. The defects were enveloped in sleeves composed of nonbiodegradable expanded polyfluoroethylene (ePTFE), pore size 30, 60, 90 microns, and compared with sleeves of three biodegradable materials. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> and associated noncollagenous <em>bone</em> matrix <em>protein</em> (BMP/NCP) or recombinant human <em>morphogenetic</em> <em>protein</em> (rhBMP-2) were implanted inside the sleeves. Albumin was implanted for a control system. Without intracompartmental BMP, only about <em>10</em>%-15% of the defect was repaired by <em>bone</em> growth extending from the <em>bone</em> ends into the sleeves composed of ePTFE, pore size 30 microns. With sleeves with pore size 60 or 90 microns and intracompartmental BMP/NCP, 54%-96% regeneration occurred within 8 weeks after the operation. Sleeves of biodegradable nonimmunogenic materials such as polyorthoester (POE) and polylactic-polyglycollic acids (PLA/PGA) permitted 86%-98% restoration of <em>bone</em> continuity, but only when BMP was present in the lumen. With puncture holes (0.5 mm in diameter), implants of BMP/NCP in the 30-micron PTFE sleeve produced transmembrane external callus formation and <em>bone</em> regeneration to 147%. Sleeves composed of aorta first calcified, then induced complete intracompartmental <em>bone</em> regeneration. Atelocollagen sleeves incited a low-grade inflammatory cell reaction and did not promote complete regeneration. Under conditions presently undisclosed segments of the ulna bridged with ePTFE, were incompletely paired, even with intracompartmental BMP/NCP. Puncture holes of 0.5 mm admitted ingrowth of capillaries and introduced local conditions favorable for the response to BMP/NCP. BMP/NCP may promote proliferation of nutrient vessels and differentiation of <em>bone</em> marrow stroma cells between the open <em>bone</em> ends. For further investigation, the hypothesis to be examined is that the optimum response to BMP/NCP and rhBMP-2 would emerge in compartments containing first a high concentration gradient and second proliferating perivascular cells.

OBJECTIVE

Tissue engineering of bone entails the successful interplay between osteoinductive factors, osteogenic cells, their extracellular environment, and an osteoconductive biomaterial scaffold. Naturally produced ceramics, like hydroxyapatite (HA) calcified from red algae, are the most promising materials for use as scaffolds in this field. We hypothesized that extracellular matrix compartments and osteoinductive factors could further ameliorate the bioactivity of the scaffold.

METHODS

Osteosarcoma cells with proven osteogenic phenotype (SaOS-2) were cultured onto type I collagen coated (Coll I/HA) and noncollagen coated HA granules (NC/HA) gained from red algae (C GRAFT/Algipore). Cells grown on tissue culture polystyrene dishes (TCPS) were used as controls. Second, SaOS-2 cells cultured on Coll I/HA, NC/HA, and TCPS were treated with recombinant human bone morphogenetic protein-2 (rhBMP-2) in different concentrations (10, 100, and 500 ng/mL). Non rhBMP-2-treated cultures were used as controls. Cultures of both experiments were grown under osteogenic differentiation conditions and after 24, 48, and 72 hours assays for cell viability, apoptosis, alkaline phosphatase activity (ALP), and osteocalcin (OC) secretion were done.

RESULTS

Coating of HA granules with type I collagen showed higher cell viability in rhBMP-2-treated and nontreated cells. Supplementation of cultured cells with exogenous rhBMP-2 showed a dose-dependent effect only in the TCPS group. No alterations of the apoptotic rate within 1 investigation group were found. Addition of rhBMP-2 did not significantly alter the specific OC secretion of cells grown on Coll I/HA and TCPS.

CONCLUSIONS

These in vitro findings show that in the initial period of cultivation and up to 72 hours, the coating of HA granules with collagen type I had positive effects on cell viability and osteoblastic characteristics of osteoblastic cells. In contrast, the supplementation with exogenous rhBMP-2 shows no dose-dependent effects. The combination of collagen type I and exogenous rhBMP-2 did not ameliorate the bioactivity of hydroxyapatite calcified from red algae in the initial period of cultivation.

Activins and <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are members of the transforming growth factor-beta family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended beta-sheet, whereas residues involved in type I binding are located in the alpha-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45-79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations <em>10</em>-fold higher than the minimal effective activin A concentration (approximately 4 nM). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-beta members, activins, and BMPs.

Depending on the type and localisation, nonunions of tibial fractures will occur in <em>10</em>-40% of cases. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP-7; Osigraft), a recombinant <em>bone</em> growth factor, can be implanted locally as an alternative to autologous <em>bone</em> grafting. The objective of our study was to compare the efficiency of the two procedures. From January 1995 to December 2002, 82 patients (group 1) with delayed union of a tibial fracture received autologous <em>bone</em> grafting as their first procedure. To compare their results with the efficiency of BMP-7, between May 2002 and June 2005 we followed up on 26 patients (group 2) who had local implantation of BMP-7 after having had, on average, four surgical procedures. Healing was considered successful if x-rays showed bony consolidation and if no further procedure was necessary. Group 1 had no signs of consolidation in 24 cases (28%), whereas group 2 had only two (8%) such patients (p=0.025). The BMP-7 group showed a significantly higher success rate compared with patients with autologous <em>bone</em> grafting, despite the fact that the BMP-7 group contained more complicated cases.

Obesity is considered a public health problem worldwide. Fenofibrate, a selective peroxisome proliferator-activated receptor α (PPAR-α) agonist, elicits weight loss in animal models. This study aimed to examine the effects of fenofibrate on energy expenditure, body mass (BM) and gene expression of thermogenic factors in brown adipose tissue of diet-induced obese mice. Male C57BL/6 mice were fed a standard chow (SC; <em>10</em>% lipids) diet or a high-fat (HF; 50% lipids) diet for <em>10</em> weeks. Afterwards, groups were subdivided as SC, SC-F, HF and HF-F (n = <em>10</em>, each). Treatment with fenofibrate (<em>10</em>0 mg kg(-1) BM mixed into the diet) lasted 5 weeks. Treated groups had reduced final BM compared with their counterparts (p < 0·05), explained by the increase in energy expenditure, CO2 production and O2 consumption after treatment with fenofibrate (p < 0·05). Similarly, genes involved in thermogenesis as PPAR-α, PPAR-γ coactivator 1α, nuclear respiratory factor 1, mitochondrial transcription factor A (Tfam), PR domain containing 16 (PRDM16), β-3 adrenergic receptor (β3-AR), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 8B and uncoupling <em>protein</em> 1 were significantly expressed in brown adipocytes after the treatment (p < 0·05). All observations ensure that selective PPAR-α agonist can induce thermogenesis by increasing energy expenditure and enhancing the expression of genes involved in the thermogenic pathway. These results suggest fenofibrate as a coadjutant drug for the treatment of obesity.

OBJECTIVE

Systemic nicotine has been hypothesized to cause degeneration of the intervertebral disc which in turn decreases vascular supply to the disc through a cholinergic receptor-mediated process. Another possible mechanism may be through direct regulatory effects on disc cells. In this study, the authors tested the hypothesis that nicotine adversely affects nucleus pulposus cells by directly inhibiting proteoglycan synthesis and gene expression of type II collagen (Phase I study). They also assessed the hypothesis that nicotine inhibits the bone morphogenetic protein (BMP)-2-induced upregulation of extracellular matrix (Phase II study).

METHODS

Cells were isolated from nucleus pulposus obtained in rat lumbar discs and cultured on a monolayer. Media were treated with nicotine and/or recombinant human (rh)BMP-2 for 7 days. Sulfated glycosaminoglycan (SO4-GAG) in media was quantified using 1,9-dimethylmethylene blue (DMMB) assay. Gene assay of types I and II collagen, Sox9, and glyceraldehyde-3-phosphate dehydrogenase were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR) and real time PCR. In the Phase I study, nicotine-treated (100 microg/ml) and non-treated cells were compared. The s-GAG production and messenger RNA (mRNA) of type II collagen and Sox9 decreased significantly in the nicotine-treated group. In the Phase II study, five groups were compared: 1) non-treatment; 2) rhBMP-2 only (100 ng/ml); and 3-5) with rhBMP-2 (100 ng/ml) and increasing doses of nicotine (1 [third group], 10, [fourth group], 100 [fifth group] microg/ml). The SO4-GAG production and mRNA of type II collagen and Sox9 decreased significantly in the groups treated with rhBMP-2 combined with 10 and 100 microg/ml of nicotine compared with the group treated with rhBMP-2.

CONCLUSIONS

The results of this study raise the possibility that nicotine may contribute to the process of disc degeneration by a direct effect on the nucleus pulposus cells, possibly by antagonizing the effect of BMP-2.

Arterial calcification is common in vascular diseases and involves conversion of vascular smooth muscle cells (VSMCs) to an osteoblast phenotype. Clinical studies suggest that the development of atherosclerosis can be promoted by homocysteine (HCY), but the mechanisms remain unclear. Here, we determined whether increases in HCY levels lead to an increase in VSMC calcification and differentiation, and examined the role of an extracellular matrix remodeler, matrix metallo<em>protein</em>ase-2 (MMP-2). Rat VSMCs were exposed to calcification medium in the absence or presence of HCY (<em>10</em>, <em>10</em>0 or 200 μmol/L) or an MMP-2 inhibitor (<em>10</em>(-6) or <em>10</em>(-5) mol/L). MTT assays were performed to determine the cytotoxicity of the MMP-2 inhibitor in calcification medium containing 200 μmol/L HCY. Calcification was assessed by measurements of calcium deposition and alkaline phosphatase (ALP) activity as well as von Kossa staining. Expression of osteocalcin, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2, and osteopontin, and MMP-2 was determined by immunoblotting. Calcification medium induced osteogenic differentiation of VSMCs. HCY promoted calcification, increased osteocalcin and BMP-2 expression, and decreased expression of osteopontin. MMP-2 expression was increased by HCY in a dose-dependent manner in VSMCs exposed to both control and calcification medium. The MMP-2 inhibitor decreased the calcium content and ALP activity, and attenuated the osteoblastic phenotype of VSMCs. Vascular calcification and osteogenic differentiation of VSMCs were positively regulated by HCY through increased/restored MMP-2 expression, increased expression of calcification <em>proteins</em>, and decreased anti-calcification <em>protein</em> levels. In summary, MMP-2 inhibition may be a protective strategy against VSMC calcification.

We have previously shown p53 to have a specific role in osteoblast differentiation by its ability to regulate expression of certain <em>bone</em> specific <em>proteins</em>. In this study, we show mineralized matrix formation in vivo to be directly related to the presence of wild type p53 in osteoblastic osteosarcoma cells. In order to further understand the importance of p53 in differentiation, we investigated the relationship between p53 and <em>Bone</em> <em>Morphogenetic</em> <em>Proteins</em> (BMPs) (BMP 1, 2, 3A, 3B (GDF-<em>10</em>), 4, 5, 6, 7, 8A and 8B) during osteoblast differentiation. The expression of several BMPs were tested using RNase Protection Assay in differentiating ROS17/2.8 osteoblastic osteosarcoma cells. The expression of BMPs 1, 2, 3a, 3b and 7 showed time dependent modulation during in vitro differentiation. In order to determine if p53 has a role in this process, we used a murine osteosarcoma cell line stably expressing a temperature sensitive p53. Cells were exposed to ascorbic acid and glycerophosphates to hasten in vitro osteoblast differentiation and maintained either at 32 or 37 degrees C for expression of the wild type or mutant p53 phenotype. The expression of BMP-2, BMP-4 and BMP-7 were modulated in a p53 dependent fashion. We were able to confirm the p53 dependency of BMP-2 independently by RT-PCR. While BMP-2 expression was evident in the presence of both wild type and mutant p53, regulated expression was seen only in cells expressing wild type p53. Transient over expression of wild type p53 did not result in the same BMP-2 response as stable expression showing that the presence of p53 may be important for an orderly development of osteoblast differentiation rather than a direct effect on gene expression. The functional relationship between p53 and these <em>bone</em> specific markers is discussed.

A composite of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) and collagen was implanted beneath the cranial periosteum of <em>10</em>-month-old rats to observe <em>bone</em> development and absorbent change of carrier collagen. The rhBMP-2/collagen onlay implant resulted in active <em>bone</em> formation and the augmented <em>bone</em> was connected directly with the original <em>bone</em>, whereas the collagen alone resulted in neither <em>bone</em> nor cartilage. The ossification process in the rhBMP-2/collagen occurred directly through <em>bone</em> formation, similar to intramembranous ossification. The carrier collagen fibers were found in the woven <em>bone</em> and were completely absorbed at 8 weeks in the presence of rhBMP-2, while the collagen alone implant remained encapsulated by a thin, fibrous connective tissue. Our results indicate that rhBMP-2/collagen is an effective material as a biological onlay implant, showing osteoinductive properties and being completely replaced by new <em>bone</em>. Carrier collagen not only plays a role in rhBMP-2 delivery, but also provides a cell anchorage for cell differentiation and remains as an artificial matrix in woven <em>bone</em>.

OBJECTIVE

The aim of this study was to evaluate the survival, proliferation, and bone formation of dog mesenchymal stem cells (dMSCs) in the graft material by using Polycaprolactone-tricalcium phosphate (PCL-TCP), auto-fibrin glue (AFG), recombinant human bone morphogenetic protein-2 (rhBMP-2), and dMSCs after a transplantation to the scapula of adult beagle dogs.

METHODS

The subjects were two beagle dogs. Total dose of rhBMP-2 on each block was 10 microg with 50 microg/mg concentration. The cortical bone of the scapula of the dog was removed which was the same size of PCL-TCP block (Osteopore International Pte, Singapore; 5.0x5.0x8.0 mm in size), and the following graft material then was fixed with orthodontic mini-implant, Dual-top (Titanium alloy, Jeil Co. Seoul, Korea). Four experimental groups were prepared for this study, Group 1: PCL-TCP + aFG; Group 2: PCL-TCP + aFG + dMSCs; Group 3: PCL-TCP + aFG + dMSCs + rhBMP-2; Group 4: PCL-TCP + aFG + dMSCs + rhBMP-2 + PCL membrane. The survival or proliferation of dMSCs cells was identified with an extracted tissue through a fluorescence microscope, H-E staining and Von-Kossa staining in two weeks and four weeks after the transplantation.

RESULTS

The survival and proliferation of dMSCs were identified through a fluorescence microscope from both Group 1 and Group 2 in two weeks and four weeks after the transplantation. Histological observation also found that the injected cells were proliferating well in the G2, G3, and G4 scaffolds.

CONCLUSIONS

This study concluded that bone ingrowth occurred in PCL-TCP scaffold which was transplanted with rhBMP-2, and MSCs did not affect bone growth. More sufficient healing time would be needed to recognize effects of dMSCs on bone formation.

We investigated ectopic <em>bone</em> formation by biphasic calcium phosphate (BCP) combined with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) in the rat dorsum. Under reduced pressure, rhBMP-2 was adsorbed onto BCP, which consisted of 80% beta-tricalcium phosphate and 20% hydroxyapatite uniformly distributed in granules. Twenty Wistar rats were separated into 4 groups consisting of 5 animals each dosed with 2, <em>10</em>, and 50 micrograms/700 microliters of rhBMP-2 and a control group (BCP only). Pieces of the BCP-BMP complex or only BCP were implanted under the dorsal skin of the rats. Histological sections were examined three weeks later. New <em>bone</em> was formed in all rats given 50 micrograms doses, but not in the 2 micrograms and control groups. These results indicated that BCP combined with rhBMP-2 induced ectopic <em>bone</em> formation without additional carriers. Therefore, BCP granules alone can function as carriers for rhBMP-2 to induce <em>bone</em> formation.

Salivary pleomorphic adenomas are often associated with chondroid tissue formation. We have found that <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP), especially BMP-2, may play an important role in ectopic chondrogenesis in this tumor. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-6 was reported to be related to the osteogenic metastasis of prostatic carcinomas. The relationship between BMP-6 expression and chondroid tissue formation is investigated. Twenty-three pleomorphic adenomas were examined immunohistochemically. The overexpression of BMP-6 was observed in <em>10</em> pleomorphic adenomas of the major salivary glands (43. 5%), and no evidence of BMP-6 expression in any of the nine pleomorphic adenomas of the palate. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-6 was immunolocalized in the lacuna cells of the chondroid tissue, in which type II collagen was localized. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-6 was expressed in inner ductal cells of the tubulo-glandular structures in the pleomorphic adenomas. This finding indicates that BMP-6 may be associated with the differentiation of inner ductal cells. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-6 was expressed weakly in neoplastic myoepithelial cells in the myxoid areas, which may be related to the production of extracellular matrices. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-6 has a role in chondroid formation, and also tubulo-glandular differentiation in pleomorphic adenomas. In conclusion, a large portion of pleomorphic adenomas of the salivary gland origin, but not of palate origin, was shown to overexpress BMP-6 <em>protein</em>.

OBJECTIVE

Recently, we reported that the forkhead transcription factor, FKHR/FOXO1, is required for vascular endothelial growth factor (VEGF)-mediated upregulation of a number of genes in endothelial cells. Here, we tested the hypothesis that hepatocyte growth factor (HGF), a potent activator of PI3K-Akt in endothelial cells, is capable of depleting the nucleus of FKHR/FOXO1 and thus inhibiting VEGF induction of this class of genes.

RESULTS

Incubation of human coronary artery endothelial cells with HGF induced prolonged PI3K/Akt-dependent phosphorylation and nuclear exclusion of FKHR/FOXO1. HGF-mediated inhibition of FKHR/FOXO1 activity resulted in secondary attenuation of VEGF-induced expression of FKHR/FOXO1-dependent genes including vascular cell adhesion molecule-1, manganese superoxide dismutase, endothelial specific molecule-1, CBP/p300 interacting transactivator with ED-rich tail-2, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2, matrix metallo<em>protein</em>ase (MMP)-<em>10</em>, and MGC5618. At a functional level, preincubation of HGF resulted in inhibition of VEGF-induced vascular cell adhesion molecule (VCAM)-1-mediated monocyte adhesion to endothelial cells. HGF-mediated inhibition of VEGF-inducible VCAM-1 expression and monocyte adhesion was reversed by overexpression of constitutively active phosphorylation-resistant triple mutant (TM)-FKHR.

CONCLUSIONS

These findings suggest that physiological agonists of PI3K-Akt signaling pathway may modulate VEGF-FKHR/FOXO1-dependent gene expression in endothelial cells. The data underscore the importance of the "set point" of the endothelial cell when considering mechanisms of signal transduction.

Pluripotent neural crest (NC) cells differentiate to diverse lineages, including the neuronal, sympathoadrenal lineage. In primary NC cultures, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) requires moderate activation of cAMP signaling for induction of the sympathoadrenal lineage. However, the mechanism by which cAMP signaling synergizes with BMP2 to induce the sympathodrenal lineage is unknown. Herein, we demonstrate that moderate activation of cAMP signaling induces both transcription and activity of proneural transcription factor Phox2a. In NC cultures inhibition of cAMP-response element-binding <em>protein</em> (CREB)-mediated transcription by expression of dominant-negative CREB suppresses Phox2a transcription and sympathoadrenal lineage development. Interestingly, the constitutively active CREB(DIEDML), despite inducing Phox2a transcription, is insufficient for sympathoadrenal lineage development, requiring activation of the cAMP pathway. Because CREB(DIEDML)-mediates cAMP-dependent transcription without requiring activation by the cAMP-dependent <em>protein</em> kinase A (PKA), these results identify PKA activation as necessary in sympathoadrenal lineage development. Treatment of NC cultures with the PKA inhibitor H89 or 1-<em>10</em> nm okadaic acid (OA), a serine/threonine PP2A-like phosphatase inhibitor, suppresses sympathoadrenal lineage development. Likewise, OA treatment of the CNS-derived catecholaminergic CAD cell line inhibits cAMP-mediated neuronal differentiation. Specifically, OA inhibits cAMP-mediated Phox2a dephosphorylation, cAMP-dependent Phox2a DNA binding in vitro, and cAMP- and Phox2a-dependent dopamine-beta-hydroxylase-luciferase reporter expression. Together, these results support cAMP-dependent Phox2a dephosphorylation is required for its activation. We conclude that moderate activation of cAMP signaling has dual inputs in catecholaminergic, sympathoadrenal lineage development; that is, regulation of both Phox2a transcription and activity. These results provide the first mechanistic understanding of how moderate activation of the cAMP pathway in synergy with BMP2 promotes sympathoadrenal lineage development.

<em>Bone</em> marrow stromal cells isolated from a model of osteogenesis imperfecta (oim) mice, were transduced with a retrovirus (BAG) carrying the LacZ and neor genes after passage 21. The transduced cells retained the ability to express alkaline phosphatase activity in vitro when treated with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> two (rhBMP-2), formed cartilage in vitro in aggregate cultures and formed <em>bone</em> in ceramic cubes after 6 weeks of implantation in nude mice. X-gal staining of ceramic cubes seeded with the transduced cells demonstrated the presence of LacZ-positive cells on the edges of <em>bone</em> and also in the lacunae of the newly formed <em>bone</em> 6 weeks after implantation. After infusion into femurs of oim mice, the transduced cells were detected in the marrow cavity and on the edges of the trabecular <em>bone</em> of the injected and contralateral femurs by X-gal staining and PCR analysis at 4, <em>10</em>, 20, 30 and 40 days after injection. The LacZ gene was also detected in the lung and liver of the recipient mice at 4 and <em>10</em> days after injection but not at later time-periods. The present findings suggest that long-term cultured <em>bone</em> marrow stromal cells from osteogenesis imperfecta (OI) animals have the potential to traffic through the circulatory system, home to <em>bone</em>, form <em>bone</em> and continue to express exogenous genes. These findings open the possibility of using these cells as vehicles to deliver normal genes to <em>bone</em> as an alternative approach for the treatment of some forms of OI and certain other <em>bone</em> acquired and genetic diseases.