Disrupted synchronized oscillatory firing of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30-<em>10</em>0 Hz) mediates many of the cognitive deficits and symptoms of schizophrenia. In fact, the density of dendritic spines and the average somal area of pyramidal neurons in layer 3 of the cerebral cortex, which mediate both long-range (associational) and local (intrinsic) corticocortical connections, are decreased in subjects with this illness. To explore the molecular pathophysiology of pyramidal neuronal dysfunction, we extracted ribonucleic acid (RNA) from laser-captured pyramidal neurons from layer 3 of Brodmann's area 42 of the superior temporal gyrus (STG) from postmortem brains from schizophrenia and normal control subjects. We then profiled the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identified 1331 mRNAs that were differentially expressed in schizophrenia, including genes that belong to the transforming growth factor beta (TGF-β) and the <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) signaling pathways. Disturbances of these signaling mechanisms may in part contribute to the altered expression of other genes found to be differentially expressed in this study, such as those that regulate extracellular matrix (ECM), apoptosis, and cytoskeletal and synaptic plasticity. In addition, we identified <em>10</em> microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of their predicted gene targets revealed signaling pathways and gene networks that were found by microarray to be dysregulated, raising an interesting possibility that dysfunction of pyramidal neurons in schizophrenia may in part be mediated by a concerted dysregulation of gene network functions as a result of the altered expression of a relatively small number of miRNAs. Taken together, findings of this study provide a neurobiological framework within which specific hypotheses about the molecular mechanisms of pyramidal cell dysfunction in schizophrenia can be formulated.

BACKGROUND

To date, no single most-appropriate factor or delivery method has been identified for the purpose of mesenchymal stem cell (MSC)-based treatment of cartilage injury. Therefore, in this study we tested whether gene delivery of the growth factor Indian hedgehog (IHH) was able to induce chondrogenesis in human primary MSCs, and whether it was possible by such an approach to modulate the appearance of chondrogenic hypertrophy in pellet cultures in vitro.

METHODS

First-generation adenoviral vectors encoding the cDNA of the human IHH gene were created by cre-lox recombination and used alone or in combination with adenoviral vectors, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (Ad.BMP-2), or transforming growth factor beta-1 (Ad.TGF-β1) to transduce human <em>bone</em>-marrow derived MSCs at 5 × <em>10</em>² infectious particles/cell. Thereafter, 3 × <em>10</em>⁵ cells were seeded into aggregates and cultured for 3 weeks in serum-free medium, with untransduced or marker gene transduced cultures as controls. Transgene expressions were determined by ELISA, and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.

RESULTS

IHH, TGF-β1 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs, as evidenced by strong staining for proteoglycans, collagen type II, increased levels of glycosaminoglycan synthesis, and expression of mRNAs associated with chondrogenesis. IHH-modified aggregates, alone or in combination, also showed a tendency to progress towards hypertrophy, as judged by the expression of alkaline phosphatase and stainings for collagen type X and Annexin 5.

CONCLUSIONS

As this study provides evidence for chondrogenic induction of MSC aggregates in vitro via IHH gene delivery, this technology may be efficiently employed for generating cartilaginous repair tissues in vivo.

Leukemia inhibitory factor plays a major role in the uterus and in its absence embryos fail to implant. Our knowledge of the targets for LIF and the consequences of its absence is still very incomplete. In this study, we have examined the ultrastructure of the potential implantation site in LIF-null MF1 female mice compared to that of wild type animals. We also compared expression of <em>proteins</em> associated with implantation in luminal epithelium and stroma. Luminal epithelial cells (LE) of null animals failed to develop apical pinopods, had increased glycocalyx, and retained a columnar shape during the peri-implantation period. Stromal cells of LIF-null animals showed no evidence of decidual giant cell formation even by day 6 of pregnancy. A number of <em>proteins</em> normally expressed in decidualizing stroma did not increase in abundance in the LIF-null animals including desmin, tenascin, Cox-2, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2 and -7, and Hoxa-<em>10</em>. In wild type animals, the IL-6 family member Oncostatin M (OSM) was found to be transiently expressed in the luminal epithelium on late day 4 and then in the stroma at the attachment site on days 5-6 of pregnancy, with a similar but not identical pattern to that of Cox-2. In the LIF-null animals, no OSM <em>protein</em> was detected in either LE or stroma adjacent to the embryo, indicating that expression requires uterine LIF in addition to a blastocyst signal. Fucosylated epitopes: the H-type-1 antigen and those recognized by lectins from Ulex europaeus-1 and Tetragonolobus purpureus were enhanced on apical LE on day 4 of pregnancy. H-type-1 antigen remained higher on day 5, and was not reduced even by day 6 in contrast to wild type uterus. These data point to a profound disturbance of normal luminal epithelial and stromal differentiation during early pregnancy in LIF-nulls. On this background, we also obtained less than a Mendelian ratio of null offspring suggesting developmental failure.

Transforming growth factors-beta (TGF-betas), activins, and <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) comprise an evolutionarily well-conserved group of <em>proteins</em> controlling a number of cell differentiation, cell growth, and morphogentic processes during development. The superfamily of TGFbeta-related genes include over 25 members in mammals several of which are expressed in the growing nervous system and serve important functions in regionalizing the early CNS. Cultured nerve cells show different responses to these factors. Recent developments have revealed that TGFbetas, activins, and BMPs selectively signal to the responding cells via different hetero-oligomeric complexes of type I and type II serine/threonine kinase receptors. The adult brain exhibits specific expression patterns of some of these receptors suggesting neuronal functions not only during development but also in the mature brain. In particular, the brain is expressing high levels of <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type II (BMPR-II), activin receptor type I (ActR-I), and activin receptor type IIA (ActR-II). This indicates that osteogenic <em>protein</em>-1 (OP-1/BMP-7), BMP-2, and BMP-4 as well as activins may serve functions for brain neurons. Expression of the receptors partially overlaps in populations of neurons and has been shown to be regulated by brain lesions. This suggests that brain neurons may use receptors BMPR-II and ActR-I to sense the presence of BMPs. This may form a system parallel to the neurotrophin Trk tyrosine kinase receptors regulating neuroplasticity and brain repair. The presence of BMPs in brain is not well studied, but preliminary in situ data indicate that the BMP relatives growth/differentiation factor (GDF)-1 and GDF-<em>10</em> are distinctly but differentially expressed at high levels in neurons expressing BMPR-II and ActR-I. The receptors mediating responses to these two GDFs remain, however, to be defined. Finally, recent data show that the signal from the activated type I serine/threonine kinase receptor is directly transduced to the nucleus by Smad <em>proteins</em> that become incorporated into transcriptional complexes. Preliminary in situ hybridization observations demonstrate the existence of different Smad mRNAs. It is concluded that BMPs and their signaling systems may comprise a novel pathway for control of neural activity and offer means for pharmacological interventions rescuing brain neurons.

Methamphetamine (MA) is a drug of abuse as well as a dopaminergic neurotoxin. We have previously demonstrated that pretreatment with <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson's disease. In this study, we examined the neuroprotective effects of BMP7 against MA-mediated toxicity in dopaminergic neurons. Primary dopaminergic neurons, prepared from rat embryonic ventral mesencephalic tissue, were treated with MA. High doses of MA decreased tyrosine hydroxylase immunoreactivity (THir) while increasing terminal deoxynucleotidyl transferase-mediated dNTP nick end labeling. These toxicities were significantly antagonized by BMP7. Interaction of BMP7 and MA in vivo was first examined in CD1 mice. High doses of MA (<em>10</em> mg/kgx4 s.c.) significantly reduced locomotor activity and THir in striatum. I.c.v. administration of BMP7 antagonized these changes. In BMP7 +/- mice, MA suppressed locomotor activity and reduced TH immunoreactivity in nigra reticulata to a greater degree than in wild type BMP7 +/+ mice, suggesting that deficiency in BMP7 expression increases vulnerability to MA insults. Since BMP7 +/- mice also carry a LacZ-expressing reporter allele at the BMP7 locus, the expression of BMP7 was indirectly measured through the enzymatic activity of beta-galactosidase (beta-gal) in BMP7 +/- mice. High doses of MA significantly suppressed beta-gal activity in striatum, suggesting that MA may inhibit BMP7 expression at the terminals of the nigrostriatal pathway. A similar effect was also found in CD1 mice in that high doses of MA suppressed BMP7 mRNA expression in nigra. In conclusion, our data indicate that MA can cause lesioning in the nigrostriatal dopaminergic terminals and that BMP7 is protective against MA-mediated neurotoxicity in central dopaminergic neurons.

METHODS

Posterolateral lumbar transverse process fusion was completed using the cultured bone marrow cells in type I collagen gel and porous hydroxyapatite.

OBJECTIVE

To compare the efficacy of cultured bone marrow cells with that of bone morphogenetic protein (BMP) as a graft alternative to autologous bone for posterolateral spine fusion.

BACKGROUND

The clinical application of BMP for spinal fusion may be limited by high dose and cost. Recently, mesenchymal stem cells have been studied in various fields because of their capability to differentiate into various cells, including those in the osteogenic lineage.

METHODS

Thirty adult rabbits were used. Each underwent single-level, bilateral, posterolateral intertransverse process fusions at L4-L5. The animals were divided into 4 groups, each according to the material implanted: (1) autologous bone (autograft, n = 9); (2) porous hydroxyapatite (HA) particles and type I collagen sheet with 100 microg rhBMP-2 (BMP-HA, n = 7); (3) bone marrow cells (1 x 10(6) cells/mL, low-marrow-HA, n = 7); and (4) bone marrow cells (1 x 10(8) cells/mL, high-marrow-HA, n = 7). Before implantation for groups 3 and 4, fresh bone marrow cells from the iliac crest of each animal were cultured in a standard medium for 2 weeks. For one additional week, the marrow cells were cultured in 10(-8) M dexamethasone, type I collagen gel, and HA. Animals were euthanized 6 weeks after surgery. Spinal fusions were evaluated by radiograph, manual palpation, and histology.

RESULTS

The fusion rates were 4 of 7 in the autograft group, 7 of 7 in the BMP-HA group, 0 of 7 in the low-marrow-HA group, and 5 of 7 in the high-marrow-HA group. The histology in the BMP-HA and high-marrow-HA groups showed that grafted HA fragments were connected with mature new bone. The pores of HA fragments were filled up with bone matrix. In the low-marrow-HA group, fibrous tissue was predominant in the grafted fragments.

CONCLUSIONS

This study shows that the cultured bone marrow cells can act as a substitute for autograft or BMP in spine fusion. The current formulation may yield improved fusion success and better quality of fusion bone as compared to autograft.

OBJECTIVE

Tooth eruption requires the presence of a dental follicle (DF), alveolar bone resorption for an eruption pathway, and alveolar bone formation at the base of the bony crypt. The objectives of our investigations have been to determine how the DF regulates both the osteoclastogenesis and osteogenesis needed for eruption.

METHODS

Multiple experimental methods have been employed.

RESULTS

The DF regulates osteoclastogenesis and osteogenesis by regulating the expression of critical genes in both a chronological and spatial fashion. In the rat 1st mandibular molar there is a major burst of osteoclastogenesis at day 3 postnatally and a minor burst at day 10. At day 3, the DF maximally expresses colony-stimulating factor-1 (CSF-1) to down-regulate the expression of osteoprotegerin (OPG) such that osteoclastogenesis can occur. At day 10, the minor burst of osteoclastogenesis is promoted by upregulation of vascular endothelial growth factor (VEGF) and RANKL in the DF. Spatially, the bone resorption is in the coronal portion of the bony crypt and genes such as RANKL are expressed more in the coronal region of the DF than in its basal one-half. For osteogenesis, bone formation begins at day 3 at the base of the bony crypt and maximal growth is at days 9-14. Osteo-inductive genes such as bone morphogenetic protein-2 (BMP-2) appear to promote this and are expressed more in the basal half of the DF than in the coronal. Conclusion - The osteoclastogenesis and osteogenesis needed for eruption are regulated by differential gene expression in the DF both chronologically and spatially.

Calcific aortic valve stenosis (CAS) is the most frequent heart valve disease in the elderly, accompanied by valve calcification. Tumor necrosis factor-α (TNF-α), a pleiotropic cytokine secreted mainly from macrophages, has been detected in human calcified valves. However, the role of TNF-α in valve calcification remains unclear. To clarify whether TNF-α accelerates the calcification of aortic valves, we investigated the effect of TNF-α on human aortic valve interstitial cells (HAVICs) obtained from patients with CAS (CAS group) and with aortic regurgitation or aortic dissection having a noncalcified aortic valve (control group). HAVICs (2 × <em>10</em>(4)) were cultured in a 12-well dish in Dulbecco's modified Eagle's medium with <em>10</em>% fetal bovine serum. The medium containing TNF-α (30 ng/ml) was replenished every 3 days after the cells reached confluence. TNF-α significantly accelerated the calcification and alkaline phosphatase (ALP) activity of HAVICs from CAS but not the control group after 12 days of culture. Furthermore, gene expression of calcigenic markers, ALP, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2), and distal-less homeobox 5 (Dlx5) were significantly increased after 6 days of TNF-α treatment in the CAS group but not the control group. Dorsomorphin, an inhibitor of mothers against decapentaplegic homologs (Smads) 1/5/8 phosphorylation, significantly inhibited the enhancement of TNF-α-induced calcification, ALP activity, Smad phosphorylation, and Dlx5 gene expression of HAVICs from the CAS group. These results suggest that HAVICs from the CAS group have greater sensitivity to TNF-α, which accelerates the calcification of aortic valves via the BMP2-Dlx5 pathway.

Recent studies have shown that alendronate (Aln) enhances the osteogenesis of osteoblasts and <em>bone</em> marrow mesenchymal stem cells. In this study, we hypothesize that Aln may act as an osteo-inductive factor to stimulate the osteogenic differentiation of human adipose-derived stem cells (hADSCs) for <em>bone</em> regeneration. The in vitro effect of Aln (1-<em>10</em> μM) on the osteogenic ability of hADSCs was evaluated by examining mineralization and alkaline phosphatase (ALP) activity. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) expression was measured using a real-time polymerase chain reaction and western blot analysis. Our results indicated that 5 μM Aln was sufficient to enhance BMP2 expression, ALP activity and mineralization in hADSCs. The in vivo effect of locally administered Aln on <em>bone</em> repair was examined in a rat critical-sized (7-mm) calvarial defect that was implanted with a hADSC-seeded poly(lactic-co-glycolic acid) (PLGA) scaffold. Aln (5 μM/<em>10</em>0 μl/day) was injected locally into the defect site for one week. New <em>bone</em> formation was evaluated by radiographic and histological analyses at 8 and 12 weeks post-implantation. The expression levels of human BMP2 (hBMP2) and hADSC localization in defect sites were examined using immunohistochemistry analysis and fluorescent in situ hybridization, respectively. Results showed that local treatment of Aln on hADSC-seeded PLGA scaffolds at week 12 had a maximal effect on <em>bone</em> regeneration, enhancing mineralization and <em>bone</em> matrix formation. In addition, hADSCs and hBMP2 were also detected at the defect sites. These results demonstrated that local delivery of Aln, a potent osteo-inductive factor, enhances hADSC osteogenesis and <em>bone</em> regeneration.

OBJECTIVE

The authors compared fusion rates in transforaminal lumbar interbody fusion (TLIFs) when using either autograft or bone morphogenetic protein (BMP) placed in the interbody space.

METHODS

Between September 2002 and December 2003, the authors performed 44 TLIF operations. Follow-up data were available for 40 patients. Of the 40 procedures, 19 involved cages filled with iliac crest autograft (Group 1) and 21 involved cages filled with a medium kit of recombinant human (rh) BMP-2 (Group 2). In all Group 2 patients, one BMP sponge was placed anterior to the cage and another was placed within the cage. In 12 of the Group 2 patients, iliac crest autograft was placed posterior to the BMP-filled cage (Group 2A). In the remaining nine Group 2 patients, only local autograft was placed posterior to the BMP-filled cage (Group 2B). Assessment of fusion was performed using dynamic radiography at 3-month intervals. Outcomes were assessed using the Prolo Scale, and iliac crest donor site pain was measured using a Visual Analog Scale (VAS). The mean follow-up period was 9 months (range 3-18 months). In Group 1 patients, one pseudarthrosis was detected. In Group 2 patients, dynamic radiography demonstrated solid fusion in all patients except one in Group 2B. Fifty-eight percent of patients in whom iliac crest autograft was used complained of donor site pain 6 months after surgery (5 of 10 points on the VAS). Symptomatic foraminal bone formation was not observed in any Group 2 patient.

CONCLUSIONS

The use of rhBMP-2 is safe in TLIFs when the sponges are placed away from the dura mater, and BMP promotes a more rapid fusion than iliac crest autograft alone. The use of rhBMP-2 in combination with local autograft is an excellent option for promoting solid fusion with TLIF, and it eliminates the possibility of iliac donor site pain.

FSH is produced in pituitary gonadotropes as an alpha/beta heterodimer, and synthesis of the beta-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHbeta can be regulated by activin and inhibin, both of which are members of the transforming growth factor-beta superfamily. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) also belong to the transforming growth factor-beta family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHbeta promoter linked to a luciferase reporter gene (oFSHbetaLuc), BMP-7 or BMP-6 was found to stimulate oFSHbetaLuc expression by 6-fold. Transient expression of the oFSHbetaLuc in a transformed gonadotrope cell line, LbetaT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by <em>10</em>- and 14-fold, respectively, from LbetaT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHbetaLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83-88% and 47-48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHbetaLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LbetaT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.

Previous studies of the direct actions of bisphosphonates on <em>bone</em> have mainly been limited to their effects on <em>bone</em>-resorbing osteoclasts and little is known about the direct effects of bisphosphonates on osteoblasts. Here we report the direct effects of alendronate on the proliferation and osteogenic differentiation of the MG-63 osteoblast-like cell line. Cell proliferation was determined with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, osteogenic differentiation was evaluated with an alkaline phosphatase bioassay and by analysis of gene expression by reverse transcription-polymerase chain reaction, and the extent of calcium deposition was measured using Alizarin Red S staining. Alendronate significantly increased cell numbers over control values, with the greatest effect at <em>10</em>(-8) M. Alkaline phosphatase activity and gene expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2, type I collagen and osteocalcin were increased after alendronate treatment. Alendronate also stimulated calcium deposition. We conclude that alendronate, apart from inhibiting osteoclastic <em>bone</em> resorption, is also a promoter of osteoblast proliferation and maturation.

Morphogenesis of the vascular system is strongly modulated by mechanical forces from blood flow. Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal-dominant disease in which arteriovenous malformations and telangiectasias accumulate with age. Most cases are linked to heterozygous mutations in Alk1 or Endoglin, receptors for <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) 9 and <em>10</em>. Evidence suggests that a second hit results in clonal expansion of endothelial cells to form lesions with poor mural cell coverage that spontaneously rupture and bleed. We now report that fluid shear stress potentiates BMPs to activate Alk1 signaling, which correlates with enhanced association of Alk1 and endoglin. Alk1 is required for BMP9 and flow responses, whereas endoglin is only required for enhancement by flow. This pathway mediates both inhibition of endothelial proliferation and recruitment of mural cells; thus, its loss blocks flow-induced vascular stabilization. Identification of Alk1 signaling as a convergence point for flow and soluble ligands provides a molecular mechanism for development of HHT lesions.

Osteogenin and related <em>bone</em> <em>morphogenetic</em> <em>proteins</em> are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and <em>bone</em> formation in vivo. The influence of osteogenin, purified from bovine <em>bone</em>, and of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor I, <em>10</em> ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.

OSF-1, more commonly known as pleiotrophin (PTN) or heparin-binding growth-associated molecule (HB-GAM), belongs to a new family of secreted HB <em>proteins</em>, which are structurally unrelated to any other growth factor family. The aims of this study were to dissect the diverse functions of PTN in <em>bone</em> formation. The study showed that PTN was synthesized by osteoblasts at an early stage of osteogenic differentiation and was present at sites of new <em>bone</em> formation, where PTN was stored in the new <em>bone</em> matrix. Low concentrations (<em>10</em> pg/ml) of PTN stimulated osteogenic differentiation of mouse <em>bone</em> marrow cells and had a modest effect on their proliferation, whereas higher concentrations (ng/ml) had no effect. However, PTN did not have the osteoinductive potential of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) because it failed to convert C2C12 cells, a premyoblastic cell line, to the osteogenic phenotype, whereas recombinant human BMP-2 (rhBMP-2) was able to do so. When PTN was present together with rhBMP-2 during the osteoinductive phase, PTN inhibited the BMP-mediated osteoinduction in C2C12 cells at concentrations between 0.05 pg/ml and <em>10</em>0 ng/ml. However, when added after osteoinduction had been achieved, PTN enhanced further osteogenic differentiation. An unusual effect of PTN (50 ng/ml) was the induction of type I collagen synthesis by chondrocytes in organ cultures of chick nasal cartilage and rat growth plates. Thus, PTN had multiple effects on <em>bone</em> formation and the effects were dependent on the concentration of PTN and the timing of its presence. To explain these multiple effects, we propose that PTN is an accessory signaling molecule, which is involved in a variety of processes in <em>bone</em> formation. PTN enhances or inhibits primary responses depending on the prevailing concentrations, the primary stimulus, and the availability of appropriate receptors.

Myocardial ischemia (MI) activates innate cardioprotective mechanisms, enhancing cardiomyocyte tolerance to ischemia. Here, we report a MI-activated liver-dependent mechanism for myocardial protection. In response to MI in the mouse, hepatocytes exhibited 6- to 19-fold upregulation of genes encoding secretory <em>proteins</em>, including α-1-acid glyco<em>protein</em> (AGP)2, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-binding endothelial regulator (BMPER), chemokine (C-X-C motif) ligand 13, fibroblast growth factor (FGF)21, neuregulin (NRG)4, proteoglycan 4, and trefoil factor (TFF)3. Five of these <em>proteins</em>, including AGP2, BMPER, FGF21, NRG4, and TFF3, were identified as cardioprotective <em>proteins</em> since administration of each <em>protein</em> significantly reduced the fraction of myocardial infarcts (37 ± 9%, 34 ± 7%, 32 ± 8%, 39 ± 6%, and 31 ± 7%, respectively, vs. 48 ± 7% for PBS at 24 h post-MI). The serum level of the five <em>proteins</em> elevated significantly in association with <em>protein</em> upregulation in hepatocytes post-MI. Suppression of a cardioprotective <em>protein</em> by small interfering (si)RNA-mediated gene silencing resulted in a significant increase in the fraction of myocardial infarcts, and suppression of all five cardioprotective <em>proteins</em> with siRNAs further intensified myocardial infarction. While administration of a single cardioprotective <em>protein</em> mitigated myocardial infarction, administration of all five <em>proteins</em> furthered the beneficial effect, reducing myocardial infarct fractions from PBS control values from 46 ± 6% (5 days), 41 ± 5% (<em>10</em> days), and 34 ± 4% (30 days) to 35 ± 5%, 28 ± 5%, and 24 ± 4%, respectively. These observations suggest that the liver contributes to cardioprotection in MI by upregulating and releasing protective secretory <em>proteins</em>. These <em>proteins</em> may be used for the development of cardioprotective agents.

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius <em>bone</em> defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for <em>10</em> days before electroporation. Mice were electroporated with either <em>bone</em> <em>morphogenetic</em> <em>protein</em> 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated <em>bone</em> formation bridging the <em>bone</em> gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion <em>bone</em> defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.

Therapeutic approaches for CKD (chronic kidney disease) have been able to reduce <em>protein</em>uria, but not diminish the disease progression. We have demonstrated beneficial effects by injection of BM (<em>bone</em> marrow)-derived MSCs (mesenchymal stem cells) from healthy donors in a rat model with CKD. However, it has recently been reported that BM-MSCs derived from uraemic patients failed to confer functional protection in a similar model. This suggests that autologous BM-MSCs are not suitable for the treatment of CKD. In the present study, we have explored the potential of MSCs derived from adipose tissue (AD-MSCs) as an alternative source of MSCs for the treatment of CKD. We have isolated AD-MSCs and evaluated their effect on the progression of CKD. Adult male SD (Sprague-Dawley) rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×<em>10</em>(6) AD-MSCs or MSC culture medium alone. The therapeutic effect was evaluated by plasma creatinine measurement, structural analysis and angiogenic/epitheliogenic <em>protein</em> expression. AD-MSCs were detected in kidney tissues from NPX animals. This group had a significant reduction in plasma creatinine levels and a lower expression of damage markers ED-1 and α-SMA (α-smooth muscle actin) (P<0.05). In addition, treated rats exhibited a higher level of epitheliogenic [Pax-2 and BMP-7 (<em>bone</em> <em>morphogenetic</em> <em>protein</em> 7)] and angiogenic [VEGF (vascular endothelial growth factor)] <em>proteins</em>. The expression of these biomarkers of regeneration was significantly related to the improvement in renal function. Although many aspects of the cell therapy for CKD remain to be investigated, we provide evidence that AD-MSCs, a less invasive and highly available source of MSCs, exert an important therapeutic effect in this pathology.

Dysregulated <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP) may contribute to the development and progression of renal cell carcinoma (RCC). Herein, we report that BMP-6 promotes the growth of RCC by interleukin (IL)-<em>10</em>-mediated M2 polarization of tumor-associated macrophages (TAM). BMP-6-mediated IL-<em>10</em> expression in macrophages required Smad5 and STAT3. In human RCC specimens, the three-marker signature BMP-6/IL-<em>10</em>/CD68 was associated with a poor prognosis. Furthermore, patients with elevated IL-<em>10</em> serum levels had worse outcome after surgery. Together, our results suggest that BMP-6/macrophage/IL-<em>10</em> regulates M2 polarization of TAMs in RCC.

The Y-box binding <em>protein</em> 1 (YB-1) is upregulated in many human malignancies including glioblastoma (GBM). It is also essential for normal brain development, suggesting that YB-1 is part of a neural stem cell (NSC) network. Here, we show that YB-1 was highly expressed in the subventricular zone (SVZ) of mouse fetal brain tissues but not in terminally differentiated primary astrocytes. Conversely, YB-1 knockout mice had reduced Sox-2, nestin, and musashi-1 expression in the SVZ. Although primary murine neurospheres were rich in YB-1, its expression was lost during glial differentiation. Glial tumors often express NSC markers and tend to loose the cellular control that governs differentiation; therefore, we addressed whether YB-1 served a similar role in cancer cells. YB-1, Sox-2, musashi-1, Bmi-1, and nestin are coordinately expressed in SF188 cells and 9/9 GBM patient-derived primary brain tumor-initiating cells (BTIC). Silencing YB-1 with siRNA attenuated the expression of these NSC markers, reduced neurosphere growth, and triggered differentiation via coordinate loss of GSK3-β. Furthermore, differentiation of BTIC with 1% serum or <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4 suppressed YB-1 <em>protein</em> expression. Likewise, YB-1 expression was lost during differentiation of normal human NSCs. Consistent with these observations, YB-1 expression increased with tumor grade (n = 49 cases). YB-1 was also coexpressed with Bmi-1 (Spearmans 0.80, P>> 0.001) and Sox-2 (Spearmans 0.66, P>> 0.001) based on the analysis of 282 cases of high-grade gliomas. These <em>proteins</em> were highly expressed in <em>10</em>/15 (67%) of GBM patients that subsequently relapsed. In conclusion, YB-1 correlatively expresses with NSC markers where it functions to promote cell growth and inhibit differentiation.

Oocyte-granulosa cell communication is essential for oocyte development. The aims of this study were: 1) to determine the effect of FSH on expression of Kit ligand (KL), growth/differentiation factor-9, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-15, and Kit during growth of oocyte-granulosa cell complexes (OGCs) in vitro; 2) to investigate the role of BMP-15 in regulation of KL expression; and 3) to correlate mRNA expression with oocyte growth. OGCs from 12-d-old mice were cultured for up to 7 d in the presence of FSH [0.05 ng/ml (low), 5 ng/ml (high)] or BMP-15 (<em>10</em> or <em>10</em>0 ng/ml). Transcripts were quantified using real-time RT-PCR, and oocyte and OGC diameters were measured. FSH regulated KL expression in a biphasic manner, with low FSH decreasing the KL-1/KL-2 ratio, and high FSH increasing the KL-1/KL-2 ratio, compared with controls (P < 0.05). The decrease in KL-1/KL-2 ratio with low FSH was due to increased KL-2 mRNA expression. Both FSH concentrations increased OGC diameter (P < 0.05), but only low FSH promoted oocyte growth (P < 0.05). High FSH also decreased BMP-15 expression (P < 0.05). FSH-stimulated oocyte growth was inhibited by Gleevec, an inhibitor of Kit activity. BMP-15 increased both KL-1 and KL-2 mRNA levels in a dose-dependent manner (P < 0.05) but did not alter the KL-1/KL-2 ratio or promote oocyte growth. When the KL-1/KL-2 ratio was increased by exogenous KL-1, FSH-stimulated oocyte growth was suppressed (P < 0.05), suggesting that lowered KL-1/KL-2 ratio is important for oocyte growth. In summary, the correct concentration of FSH is crucial for appropriate modulation of KL and BMP-15 to promote oocyte growth.

Predictable pulp capping procedures remain problematic, possibly because of the lack of appropriate stimulating factors for dentin formation. The present study examines the ability of one such stimulating factor, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2, to accelerate the differentiation of human dental pulp cells into odontoblasts. The number and morphology of cells between groups treated with 0 and <em>10</em>0 ng/ml of human recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) did not significantly differ. However, ALPase activity (a marker for biomineralization) in the group stimulated with rhBMP-2 was more than double that of the control group. We then measured the expression of mRNA encoding dentin sialophospho<em>protein</em> (DSPP) as a marker of odontoblasts in rhBMP-2-stimulated human pulp cells using a quantitative polymerase chain reaction. The expression of DSPP mRNA in cells stimulated for 24 h by <em>10</em>00 ng/ml of rhBMP-2 was approximately 20-fold and 5-fold higher than that by stimulated by <em>10</em> and <em>10</em>0 ng/ml, respectively. These findings show that rhBMP-2 promoted the differentiation of human dental pulp cells into odontoblasts but did not affect cell proliferation, suggesting that rhBMP-2 may have therapeutic utility in vital pulp therapy.

BACKGROUND

Most dental implants are positioned using a drilling surgery technique. However, dentistry recently experienced the implementation of piezoelectric surgery. This technique was introduced to overcome some of the limitations involving rotating instruments in bone surgery. This study used biomolecular and histologic analyses to compare the osseointegration of porous implants positioned using traditional drills versus the piezoelectric bone surgery technique.

METHODS

Porous titanium implants were inserted into minipig tibias. Histomorphology and levels of bone morphogenetic protein (BMP)-4, transforming growth factor (TGF)-beta2, tumor necrosis factor-alpha, and interleukin-1beta and -10 were evaluated in the peri-implant osseous samples.

RESULTS

Histomorphological analyses demonstrated that more inflammatory cells were present in samples from drilled sites. Also, neo-osteogenesis was consistently more active in bone samples from the implant sites that were prepared using piezoelectric bone surgery. Moreover, bone around the implants treated with the piezoelectric bone surgery technique showed an earlier increase in BMP-4 and TGF-beta2 proteins as well as a reduction in proinflammatory cytokines.

CONCLUSIONS

Piezoelectric bone surgery appears to be more efficient in the first phases of bone healing; it induced an earlier increase in BMPs, controlled the inflammatory process better, and stimulated bone remodeling as early as 56 days post-treatment.

BACKGROUND

Pulmonary arterial hypertension is characterized by endothelial dysregulation, but global changes in gene expression have not been related to perturbations in function.

OBJECTIVE

RNA sequencing was used to discriminate changes in transcriptomes of endothelial cells cultured from lungs of patients with idiopathic pulmonary arterial hypertension versus control subjects and to assess the functional significance of major differentially expressed transcripts.

METHODS

The endothelial transcriptomes from the lungs of seven control subjects and six patients with idiopathic pulmonary arterial hypertension were analyzed. Differentially expressed genes were related to bone morphogenetic protein type 2 receptor (BMPR2) signaling. Those down-regulated were assessed for function in cultured cells and in a transgenic mouse.

RESULTS

Fold differences in 10 genes were significant (P < 0.05), four increased and six decreased in patients versus control subjects. No patient was mutant for BMPR2. However, knockdown of BMPR2 by siRNA in control pulmonary arterial endothelial cells recapitulated 6 of 10 patient-related gene changes, including decreased collagen IV (COL4A1, COL4A2) and ephrinA1 (EFNA1). Reduction of BMPR2-regulated transcripts was related to decreased β-catenin. Reducing COL4A1, COL4A2, and EFNA1 by siRNA inhibited pulmonary endothelial adhesion, migration, and tube formation. In mice null for the EFNA1 receptor, EphA2, versus control animals, vascular endothelial growth factor receptor blockade and hypoxia caused more severe pulmonary hypertension, judged by elevated right ventricular systolic pressure, right ventricular hypertrophy, and loss of small arteries.

CONCLUSIONS

The novel relationship between BMPR2 dysfunction and reduced expression of endothelial COL4 and EFNA1 may underlie vulnerability to injury in pulmonary arterial hypertension.