Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in either control medium or medium supplemented with 1, <em>10</em>, or <em>10</em>0 ng/mL of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) BMP-2, BMP-12, or BMP-13. Under all conditions investigated, cell-polymer constructs cultivated for 4 weeks in vitro macroscopically and histologically resembled native cartilage. Addition of <em>10</em>0 ng/mL of BMP-2, BMP-12, or BMP-13 increased the total mass of the constructs relative to the controls by 121%, 80%, and 62%, respectively, which was accompanied by increases in the absolute amounts of collagen, glycosaminoglycans (GAG), and cells. The addition of <em>10</em>0 ng/mL of BMP-2, BMP-12, or BMP-13 increased the weight percentage of GAG in the constructs by 27%, 18%, and 15%, and decreased the weight percent of total collagen to 63%, 89%, and 83% of controls, respectively. BMP-2, but not BMP-12 or BMP-13 promoted chondrocyte hypertrophy. Taken together, these data suggest that BMP-2, BMP-12, and BMP-13 increase growth rate and modulate the composition of engineered cartilage and that <em>10</em>0 ng/mL of BMP-2 has the greatest effect. In addition, in vitro engineered cartilage provides a system for studying the effects of BMPs on chondrogenesis in a well-defined environment.

OBJECTIVE

To determine the relative level and localization of bone morphogenetic protein (BMP-4 mRNA in the retina and retinal pigmented epithelium (RPE) under normal and pathologic conditions, to seek clues regarding possible functions.

METHODS

Clones isolated from an RPE cDNA library were sequenced and used as probes for northern blot analysis. Expression in the retina and RPE was investigated in mouse models using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. The effect of recombinant proteins on RPE proliferation was investigated by thymidine incorporation.

RESULTS

Bovine clones with high homology to BMP-2 and BMP4 were isolated from a subtracted RPE cDNA library. Northern blot analysis using the clones as probes demonstrated abundant and differential expression in adult bovine RPE, but with RT-PCR and in situ hybridization, expression was also demonstrated in mouse retinal neurons. In mice with oxygen-induced ischemic retinopathy there was a striking decrease in BMP-4 mRNA in the retina within 6 hours of the onset of hypoxia that was maintained for at least 5 days. In mice with inherited photoreceptor degeneration, there was a dramatic decrease in BMP4 mRNA in retina and RPE during and after the degeneration. mRNA for the type II BMP receptor was observed in freshly isolated and cultured RPE cells, isolated retina, and freshly isolated bovine aortic endothelial cells. Thymidine incorporation in early-passage RPE cells showed a 14-fold stimulation above control with 5% serum that was decreased to 322%, 393%, and 313% in the presence of BMP-2 (10 ng/ml), BMP4 (10 ng/ml), and transforming growth factor (TGF)-,1 (2 ng/ml), respectively.

CONCLUSIONS

BMP-2 and BMP-4 may serve as negative growth regulators in the retina and RPE that are downregulated by injury, to allow tissue repair. Modulation of expression of the BMPs may provide a means to control the exaggerated wound repair that occurs in proliferative retinopathies.

Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to <em>10</em>% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12.

It is known that <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP-4) has a diverse effect on ESCs. However, its precise mechanism in mouse ESCs is not fully understood. We evaluated the effect of BMP-4 on ESC proliferation and its related signal cascades in this study. BMP-4 significantly increased the level of [(3)H]-thymidine incorporation in time- >> or =8 hours) and dose- >> or =<em>10</em> ng/ml) dependent manners. Additionally, BMP-4 increased cyclin D1 and decreased p27(kip1) expression values in a time-dependent manner. The increases in BMP-4-induced [(3)H]-thymidine incorporation and cyclin D1 expression were inhibited by the BMP-4 receptor antagonist noggin. BMP-4 increased Wnt1 expression. Wnt1 expression was attenuated by Smad4 small interfering RNA (siRNA), and BMP-4-induced cyclin D1 expression was inhibited by Smad4 and Wnt1 siRNAs. BMP-4 also activated beta-catenin, which was blocked by Smad4 and Wnt1 siRNAs. In addition, BMP-4 induced Akt phosphorylation. BMP-4-induced beta-catenin activation and cyclin D1 expression were attenuated by phosphatidyl inositol 3-kinase (PI3K) siRNA and Akt inhibitor. Additionally, downregulation of Smad4, Wnt1, and PI3K expression by siRNA decreased the levels of pluripotency marker mRNAs of ESCs, including Oct4, Sox2, and FoxD3. Our results suggested that BMP-4-induced [(3)H]-thymidine incorporation was significantly attenuated by Smad4, Wnt1, and PI3K knockdown. In conclusion, BMP-4 contributed to the maintenance of cell proliferation and the pluripotent state by Smad, PI3K/Akt, and Wnt1/beta-catenin in mouse ESCs.

Transplantation of <em>bone</em> marrow-derived mesenchymal stem cells (BMSCs) can repair acute kidney injury (AKI), but with limited effect. We test the hypothesis that CXCR4 overexpression improves the repair ability of BMSCs and that this is related to increased homing of BMSCs and increased release of cytokines. Hypoxia/reoxygenation-pretreated renal tubular epithelial cells (HR-RTECs) were used. BMSCs, null-BMSCs, and CXCR4-BMSCs were cocultured with HR-RTECs. The number of migrating BMSCs was counted. Proliferating cell nuclear antigen (PCNA) expression, cell death, and expressions of cleaved caspase-3 and Bcl-2 in cocultured HR-RTECs were measured. Cytokeratin 18 (CK18) expression and cytokine secretions of the BMSCs cultured with HR-RTEC supernatant were detected. BMSC homing, renal function, proliferation, and cell death of tubular cells were assayed in the AKI mouse model. CXCR4-BMSCs showed a remarkable expression of CXCR4. Stromal cell-derived factor-1 in the HR-RTEC supernatant was increased. Migration of BMSCs was CXCR4-dependent. Proportions of CK18(+) cells in BMSCs, null-BMSCs, and CXCR4-BMSCs showed no difference. However, CXCR4 overexpression in BMSCs stimulated secretion of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7, hepatocyte growth factor, and interleukin <em>10</em>. The neutralizing anti-CXCR4 antibody AMD3<em>10</em>0 abolished this. In cocultured HR-RTECs the proportions of PCNA(+) cells and Bcl-2 expression were enhanced; however, the proportion of annexin V(+) cells and expression of cleaved caspase-3 were reduced. The in vivo study showed increased homing of CXCR4-BMSCs in kidneys, which was associated with improved renal function, reduced acute tubular necrosis scoring, accelerated mitogenic response of tubular cells, and reduced tubular cell death. The enhanced homing and paracrine actions of BMSCs with CXCR4 overexpression suggest beneficial effects of such cells in BMSC-based therapy for AKI.

In <em>bone</em> tissue engineering, growth factors are widely used. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) and vascular endothelial growth factor (VEGF) are the most well-known regulators of osteogenesis and angiogenesis. We investigated whether the timing of dual release of VEGF and BMP-2 influences the amount of <em>bone</em> formation in a large-animal model. Poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were loaded with BMP-2 or VEGF to create sustained-release profiles, and rapidly degrading gelatin was loaded with either growth factor for fast-release profiles. To study in vivo osteogenicity, the two delivery vehicles were combined with biphasic calcium phosphate (BCP) scaffolds and implanted in <em>10</em> Beagle dogs for 9 weeks, at both ectopic (paraspinal muscles) and orthotopic sites (critical-size ulnar defect). The 9 ectopic groups contained combined or single BMP/VEGF dosage, in sustained- or fast-release profiles. In the ulnae of 8 dogs, fast VEGF and sustained BMP-2 were applied to one leg, and the other received the opposite release profiles. The two remaining dogs received bilateral control scaffolds. <em>Bone</em> growth dynamics was analyzed by fluorochrome injection at weeks 3, 5, and 7. Postoperative and posteuthanization X-rays of the ulnar implants were taken. After 9 weeks of implantation, <em>bone</em> quantity and <em>bone</em> growth dynamics were studied by histology, histomorphometry, and fluorescence microscopy. The release of the growth factors resulted in both enhanced orthotopic and ectopic <em>bone</em> formation. <em>Bone</em> formation started before 3 weeks and continued beyond 7 weeks. The ectopic BMP-2 fast groups showed significantly more <em>bone</em> compared to sustained release, independent of the VEGF profile. The ulna implants revealed no significant differences in the amount of <em>bone</em> formed. This study shows that timing of BMP-2 release largely determines speed and amount of ectopic <em>bone</em> formation independent of VEGF release. Furthermore, at the orthotopic site, no significant effect on <em>bone</em> formation was found from a timed release of growth factors, implicating that timed-release effects are location dependent.

BACKGROUND

Retrograde ejaculation (RE) is a complication of anterior lumbar interbody fusion (ALIF) techniques. Most commonly, this results from mechanical or inflammatory injury to the superior hypogastric plexus near the aortic bifurcation. Bone morphogenetic protein-2 (BMP-2) has been used in spinal fusions and has been associated with inflammatory and neuroinflammatory adverse reactions, which may contribute to RE development after anterior lumbar surgery.

OBJECTIVE

While controlling for anterior approach technique, we compared the incidence of RE with and without rhBMP-2 exposure, in large, matched cohorts of patients after ALIF.

METHODS

Retrospective analysis of 10 years of prospectively gathered outcomes data on consecutive-patient cohorts having the same anterior exposure technique for ALIF with and without rhBMP-2 use.

METHODS

All male patients without baseline sexual incapacity and having ALIF for lumbar spondylosis or spondylolisthesis of the lowest one or two lumbar levels with and without rhBMP-2, from 2002 through 2011.

METHODS

Diagnosis of RE as a new finding after ALIF compared against BMP-2 exposure, comorbid conditions, and other urological complications after ALIF surgery.

METHODS

From the comprehensive surgical database at a high volume, university practice, male subjects having ALIF at one (L5/S1) or two levels (L4/5, L5/S1) from 2002 to 2011 were identified. Baseline comorbid factors, postoperative urinary catheter/retention events, and RE events were recorded and comparative incidence compared.

RESULTS

There were four consecutive-patient cohorts identified: one before rhBMP-2 use was adopted (n=174), two cohorts in which BMP-2 use was routine (n=88 and n=151), and one final cohort after BMP-2 use was discontinued from routine use (n=59). The cohorts with and without BMP-2 exposure were closely comparable for age, approach, levels of surgery, comorbid factors affecting RE. Of 239 patients with ALIF and exposure to BMP-2, RE was diagnosed in 15 subjects (6.3%), compared with an RE diagnosis rate of two of 233 control patients without BMP-2 exposure (0.9%; p=.0012). Urinary retention after bladder catheter removal was also more frequently observed in patients exposed to BMP-2 (9.7%) compared with control patients (4.6%; p=.043). Of the baseline comorbid factors, medical or surgical treatment for prostatic hypertrophy disease was associated with an increased risk of RE in the BMP-2 patients (p=.034).

CONCLUSIONS

This study confirms previous reports of a higher rate of RE in ALIF procedures using rhBMP-2 and an open anterior approach to the spine. This effect may be associated with an increased risk of postoperative urinary retention after BMP-2 exposure. The magnitude of the RE effect may be increased with concomitant prostatic disease treatments.

Maternal administration of dexamethasone (DEX) for 48 h early in rat kidney development results in offspring with a reduced nephron endowment. However, the mechanism through which DEX inhibits nephrogenesis is unknown. In this study, we hypothesized that DEX may indirectly inhibit nephrogenesis by inhibiting ureteric branching morphogenesis. Whole metanephroi from embryonic day 14.5 (E14.5) rat embryos were cultured in the presence of DEX. DEX (<em>10</em>(-5) M) exposure for 2 days significantly inhibited ureteric branching compared with metanephroi grown in control media or DEX (<em>10</em>(-7) M). Culturing metanephroi for a further 3 days (in control media only) reduced total glomerular number in metanephroi previously exposed to DEX (<em>10</em>(-5) M) or (<em>10</em>(-7) M) compared with control cultures. Expression of genes known to regulate ureteric branching morphogenesis was determined by real-time PCR in metanephroi after 2 days in culture. DEX exposure in vitro decreased expression of glial cell line-derived neurotrophic factor (GDNF) and increased expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4 (BMP-4) and transforming growth factor-beta1 (TGF-beta1). Similar gene expression changes were found in E16.5 metanephroi in which the dam had been exposed to 2 days of DEX (0.2 mg.kg(-1).day(-1)) at E14.5/15.5 in vivo. However, in kidneys collected at E20.5 after in vivo exposure for 2 days, GDNF expression was increased and BMP-4 and TGF-beta1 expression decreased suggesting a biphasic response in gene expression to DEX exposure. These results show for the first time that inhibition of ureteric branching morphogenesis may be a key mechanism through which DEX exposure results in a reduced nephron endowment.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em>-6 (BMP-6) belongs to the family of TGF-beta-related growth factors. In the developing epidermis, expression of BMP-6 coincides with the onset of stratification. Expression persists perinatally but declines after day 6 postpartum, although it can still be detected in adult skin by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. We constitutively overexpressed BMP-6 in suprabasal layers of interfollicular epidermis in transgenic mice using a keratin <em>10</em> promoter. All mice expressing the transgene developed abnormalities in the skin, indicating an active transgene-derived factor. Depending on the pattern of transgene expression, the effects on proliferation and differentiation were completely opposite. Strong and uniform expression of the BMP-6 transgene resulted in severe repression of cell proliferation in embryonic and perinatal epidermis but had marginal effects on differentiation. Weaker and patchy expression of the transgene evoked strong hyperproliferation and parakeratosis in adult epidermis and severe perturbations of the usual pattern of differentiation. These perturbations included changes in the expression of keratins and integrins. Together with an inflammatory infiltrate both in the dermis and in the epidermis, these aspects present all typical histological and biochemical hallmarks of a human skin disease: psoriasis.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are potential regulators of prostate cancer cell growth and metastasis that signal through an interaction with BMP membrane receptors (BMPRs) type I and type II. In the present study, Western blot and immunohistochemical analysis of BMPRs were carried out in benign and malignant human prostate tissues to explain the loss of BMP response in human prostate cancer cells. The results demonstrated that the benign prostate specimens expressed high levels of all three BMPRs. In normal prostate, BMPRs were localized predominantly to epithelial cells. Among prostate cancer specimens, well-differentiated cancers were positive for the expression of BMPR-II, BMPR-IA, and BMPR-IB, for the most part. In contrast, only 1 of <em>10</em> poorly differentiated prostate cancer cases was positive for each of the three BMPRs (P < 0.005 for all three receptors). Taken together, these results indicate that human prostate cancer cells frequently exhibit loss of expression of BMPRs and suggest that loss of BMPRs may play an important role during the progression of prostate cancer.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are morphogens implicated in embryonic and regenerative odontogenic differentiation. Gene therapy has the potential to induce reparative dentin formation for potential pulp capping. We have optimized the gene transfer of Growth/differentiation factor 11 (Gdf11)/Bmp11 plasmid DNA into dental pulp stem cells by sonoporation in vivo. Dental pulp tissue treated with plasmid pEGFP or CMV-LacZ in 5-<em>10</em>% Optison (Molecular Biosystems Inc., San Diego, CA) and stimulated by ultrasound (1 MHz, 0.5 W/cm(2), 30 sec) showed significant efficiency of gene transfer and high level of <em>protein</em> production selectively in the local region, within 500 microm of the amputated site of the pulp tissue. The Gdf11 cDNA plasmid transferred into dental pulp tissue by sonoporation in vitro, induced the expression of dentin sialo<em>protein</em> (Dsp), a differentiation marker for odontoblasts. The transfection of Gdf11 by sonoporation stimulated a large amount of reparative dentin formation on the amputated dental pulp in canine teeth in vivo. These results suggest the possible use of BMPs using ultrasound-mediated gene therapy for endodontic dental treatment.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and <em>bone</em>-inductive <em>proteins</em> derived from <em>bone</em> matrix and belongs to the transforming growth factor beta (TGF-beta) superfamily. These <em>bone</em>-inductive <em>proteins</em> isolated from adult <em>bone</em> may be involved in <em>bone</em> repair. However, they may also play a role in cartilage and <em>bone</em> formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of <em>10</em> ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of <em>10</em> ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor beta isoforms (TGF-beta 1 and TGF-beta 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGF beta was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-beta isoforms.

BACKGROUND

Previous studies indicate that patients with pulmonary arterial hypertension (PAH) carrying a mutation in the <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type 2 (BMPR2) gene, develop the disease <em>10</em> years earlier than non-carriers, and have a more severe hemodynamic compromise at diagnosis. A recent report has suggested that this may only be the case for females and that patients with missense mutations in BMPR2 gene have more severe disease than patients with truncating mutations.

METHODS

We reviewed data from all patients with PAH considered as idiopathic and patients with a family history of PAH, who underwent genetic counselling in the French PAH network between January, 1st 2004 and April, 1st 20<em>10</em>. We compared clinical, functional, and hemodynamic characteristics between carriers and non-carriers of a BMPR2 mutation, according to gender or BMPR2 mutation type.

RESULTS

PAH patients carrying a BMPR2 mutation (n = 115) were significantly younger at diagnosis than non-carriers (n = 267) (35.8 +/- 15.4 and 47.5 +/- 16.2 respectively, p < 0.0001). The presence of a BMPR2 mutation was associated with a younger age at diagnosis in females (36.4 +/- 14.9 in BMPR2 mutation carriers and 47.4 +/- 15.8 in non-carriers, p < 0.0001), and males (34.6 +/- 16.8 in BMPR2 mutation carriers and 47.8 +/- 17.1 in non-carriers, p < 0.0001). BMPR2 mutation carriers had a more severe hemodynamic compromise at diagnosis, but this was not influenced by gender. No differences in survival and time to death or lung transplantation were found in male and female PAH patients carrying a BMPR2 mutation. No differences were observed in clinical outcomes according to the type of BMPR2 mutations (missense, truncating, large rearrangement or splice defect).

CONCLUSIONS

When compared to non-carriers, BMPR2 mutation carriers from the French PAH network are younger at diagnosis and present with a more severe hemodynamic compromise, irrespective of gender. Moreover, BMPR2 mutation type had no influence on clinical phenotypes in our patient population.

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or <em>10</em>% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. <em>10</em>% group, 1% vs. control, <em>10</em>% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Chronic kidney disease (CKD) is a major public health issue. At the histological level, renal fibrosis is the final common pathway of progressive kidney disease irrespective of the initial injury. Considerable evidence now indicates that renal inflammation plays a central role in the initiation and progression of CKD. Some of the inflammatory signaling molecules involved in CKD include: monocyte chemoattractant <em>protein</em>-1 (MCP-1), bradykinin B1 receptor (B1R), nuclear factor κB (NF-κB), tumor necrosis factor-α (TNFα), transforming growth factor β (TGF-β), and platelet-derived growth factor (PDGF). Multiple antifibrotic factors, such as interleukin-<em>10</em> (IL-<em>10</em>), interferon-γ (IFN-γ), <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7), hepatocyte growth factor (HGF) are also downregulated in CKD. Therefore, restoration of the proper balance between pro- and antifibrotic signaling pathways could serve as a guiding principle for the design of new antifibrotic strategies that simultaneously target many pathways. The purpose of this review is to summarize the existing body of knowledge regarding activation of cytokine pathways and infiltration of inflammatory cells as a starting point for developing novel antifibrotic therapies to prevent progression of CKD.

OBJECTIVE

Adipose stromal cells and dissociated brown adipose tissue have been shown to generate cardiomyocyte-like cells. However, it is not clear whether white mature adipocytes have the same potential, even though a close relationship has been found between adipocytes and vascular endothelial cells, another cardiovascular cell type. The objective of this study was to examine if white adipocytes would be able to supply cardiomyocytes.

RESULTS

We prepared a highly purified population of lipid-filled adipocytes from mice, 6-7 weeks of age. When allowed to lose lipids, the adipocytes assumed a fibroblast-like morphology, so-called dedifferentiated fat (DFAT) cells. Subsequently, <em>10</em>-15% of the DFAT cells spontaneously differentiated into cardiomyocyte-like cells, in which the cardiomyocyte phenotype was identified by morphological observations, expression of cardiomyocyte-specific markers, and immunocytochemical staining. In addition, electrophysiological studies revealed pacemaker activity in these cells, and functional studies showed that a beta-adrenergic agonist stimulated the beating rate, whereas a beta-antagonist reduced it. In vitro treatment of newly isolated adipocytes or DFAT cells with inhibitors of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP) and Wnt signalling promoted the development of the cardiomyocyte phenotype as determined by the number or beating colonies of cardiomyocyte-like cells and expression of troponin I, a cardiomyocyte-specific marker. Inhibition of BMP was most effective in promoting the cardiomyocyte phenotype in adipocytes, whereas Wnt-inhibition was most effective in DFAT cells.

CONCLUSIONS

White mature adipocytes can differentiate into cardiomyocyte-like cells, suggesting a link between adipocyte and cardiomyocyte differentiation.

BACKGROUND

Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This results in both increase in pulmonary arterial pressure and pulmonary vascular resistance. Recent studies have shown the implication of the signal transducer and activator of transcription 3 (STAT3)/bone morphogenetic protein receptor 2 (BMPR2)/peroxisome proliferator-activated receptor gamma (PPARγ) in PAH. STAT3 activation induces BMPR2 downregulation, decreasing PPARγ, which both contribute to the proproliferative and antiapoptotic phenotype seen in PAH. In chondrocytes, activation of this axis has been attributed to the advanced glycation end-products receptor (RAGE). As RAGE is one of the most upregulated proteins in PAH patients' lungs and a strong STAT3 activator, we hypothesized that by activating STAT3, RAGE induces BMPR2 and PPARγ downregulation, promoting PAH-PASMC proliferation and resistance to apoptosis.

RESULTS

In vitro, using PASMCs isolated from PAH and healthy patients, we demonstrated that RAGE is overexpressed in PAH-PASMC (6-fold increase), thus inducing STAT3 activation (from 10% to 40% positive cells) and decrease in BMPR2 and PPARγ levels (>50% decrease). Pharmacological activation of RAGE in control cells by S100A4 recapitulates the PAH phenotype (increasing RAGE by 6-fold, thus activating STAT3 and decreasing BMPR2 and PPARγ). In both conditions, this phenotype is totally reversed on RAGE inhibition. In vivo, RAGE inhibition in monocrotaline- and Sugen-induced PAH demonstrates therapeutic effects characterized by PA pressure and right ventricular hypertrophy decrease (control rats have an mPAP around 15 mm Hg, PAH rats have an mPAP >40 mm Hg, and with RAGE inhibition, mPAP decreases to 20 and 28 mm Hg, respectively, in MCT and Sugen models). This was associated with significant improvement in lung perfusion and vascular remodeling due to decrease in proliferation (>50% decrease) and BMPR2/PPARγ axis restoration (increased by ≥60%).

CONCLUSIONS

We have demonstrated the implications of RAGE in PAH etiology. Thus, RAGE constitutes a new attractive therapeutic target for PAH.

The aim of this study was to investigate the odontogenic differentiation of human dental pulp stem cells (DPSCs) on nanofibrous (NF)-poly(l-lactic acid) (PLLA) scaffolds in vitro and in vivo. Highly porous NF-PLLA scaffolds which mimic the architecture of collagen type I fibers were fabricated by the combination of a phase-separation technique and a porogen-leaching method. The human DPSCs were then seeded onto the scaffolds and cultured in different media for odontogenic differentiation: "Control" medium without supplements; "DXM" medium containing <em>10</em>(-8)M dexamethasone (DXM), 50 microgml(-1) ascorbic acid and 5mM beta-glycerophosphate; "BMP-7+DXM" medium containing <em>10</em>(-8)M DXM, 50 microgml(-1) ascorbic acid, 5mM beta-glycerophosphate plus 50 ngml(-1) <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP-7). For odontogenic differentiation study in vitro, alkaline phosphatase activity quantification, reverse transcription polymerase chain reaction, scanning electron microscopy, von Kossa staining and calcium content quantification were carried out. While both "DXM" medium and "BMP-7+DXM" medium induced the DPSCs to odontoblast-like cells, the "BMP-7+DXM" medium had greater inducing capacity than the "DXM" medium. Consistent with the in vitro studies, the "BMP-7+DXM" group presented more extracellular matrix and hard tissue formation than the "DXM" group after 8 weeks of ectopic implantation in nude mice. Differentiation of DPSCs into odontoblast-like cells was identified by the positive immunohistochemical staining for dentin sialo<em>protein</em>. In conclusion, odontogenic differentiation of DPSCs can be achieved on NF-PLLA scaffolds both in vitro and in vivo; the combination of BMP-7 and DXM induced the odontogenic differentiation more effectively than DXM alone. The NF-PLLA scaffold and the combined odontogenic inductive factors provide excellent environment for DPSCs to regenerate dental pulp and dentin.

The members of the transforming growth factor β (TGF-β) superfamily are essential regulators of cell differentiation, phenotype and function, and have been implicated in the pathogenesis of many diseases. Myocardial infarction is associated with induction of several members of the superfamily, including TGF-β1, TGF-β2, TGF-β3, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2, BMP-4, BMP-<em>10</em>, growth differentiation factor (GDF)-8, GDF-11 and activin A. This manuscript reviews our current knowledge on the patterns and mechanisms of regulation and activation of TGF-β superfamily members in the infarcted heart, and discusses their cellular actions and downstream signaling mechanisms. In the infarcted heart, TGF-β isoforms modulate cardiomyocyte survival and hypertrophic responses, critically regulate immune cell function, activate fibroblasts, and stimulate a matrix-preserving program. BMP subfamily members have been suggested to exert both pro- and anti-inflammatory actions and may regulate fibrosis. Members of the GDF subfamily may also modulate survival and hypertrophy of cardiomyocytes and regulate inflammation. Important actions of TGF-β superfamily members may be mediated through activation of Smad-dependent or non-Smad pathways. The critical role of TGF-β signaling cascades in cardiac repair, remodeling, fibrosis, and regeneration may suggest attractive therapeutic targets for myocardial infarction patients. However, the pleiotropic, cell-specific, and context-dependent actions of TGF-β superfamily members pose major challenges in therapeutic translation.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are members of the transforming growth factor-beta superfamily and signal through a number of membrane receptors. We have previously demonstrated that the loss of expression of BMP receptors (BMPRs) type IA, -IB, and -II (BMP-RIA, -RIB, and -RII) correlates with Gleason score in prostate cancer patients. To evaluate the prognostic value of this observation, we used immunohistochemistry to investigate the expression of BMPRs in association with disease progression in 60 patients. The results demonstrated a significant association between the loss of expression of the three BMPRs and Gleason score and clinical stage. However, only the loss of expression of BMP-RII showed a statistically significant association with 5-year survival rate (P<0.05) and biochemical recurrence-free rate following radical prostatectomy (P<0.005). To elucidate the effect of an abnormal BMP signaling in prostate cancer cells, we transfected dominant-negative BMP-RII (BMP-RIIDN) into the human prostate cancer cell line, PC3M. When a stable clone overexpressing BMP-RIIDN was inoculated subcutaneously into nude mice, the tumor growth rate was approximately <em>10</em> times that of control and parental cell line. These observations, taken together, indicate that the loss of BMP-RII expression as measured by immunohistochemistry may be a prognostic marker in prostate cancer patients, and that the loss of BMP-RII function may result in increased tumorigenicity in human prostate cancer cells.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient <em>bone</em> formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs) by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with <em>10</em>-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH), chondroitin sulfate (CS), and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in <em>bone</em> regeneration.

The propensity for breast cancer cells to metastasize to <em>bone</em> and to induce osteolysis has long been recognized. Characteristics of both the tumor cells and the <em>bone</em> microenvironment contribute to this phenomenon. The presence of tumor in <em>bone</em> is associated with activation of osteoclasts, resulting in excessive <em>bone</em> resorption and subsequent osteolysis. Breast cancer cells and other tumor types influence osteoclastic <em>bone</em> resorption by increasing the number of osteoclasts and enhancing their resorptive activity. Parathyroid hormone-related peptide, in addition to its role in humorally mediated hypercalcemia, is secreted by metastatic breast cancer cells in <em>bone</em> in which it acts as a paracrine factor to stimulate osteoclasts. As <em>bone</em> matrix is broken down by activated osteoclasts, a rich supply of mitogenic factors is released, including insulin-like growth factors, <em>bone</em> <em>morphogenetic</em> <em>proteins</em>, and fibroblast growth factors. Transforming growth factor (TGF)-beta, one of the most abundant of the <em>bone</em>-derived factors, promotes increased production of parathyroid hormone-related peptide by tumor cells, establishing a "vicious cycle" leading to progressive tumor growth and <em>bone</em> destruction. Bisphosphonates interrupt this cycle by inhibiting osteoclasts, in part by inducing osteoclast apoptosis. In several animal models of breast cancer metastasis to <em>bone</em>, bisphosphonates decrease the number of new <em>bone</em> metastases and inhibit progression of existing lesions. A single 3 microg intravenous injection of zoledronic acid (Zometa; Novartis Pharmaceuticals Corp, East Hanover, NJ), a new highly potent bisphosphonate, prevented destruction of trabecular <em>bone</em> in an orthotopic mouse mammary tumor model. Tumor volume in <em>bone</em> was decreased by zoledronic acid in a dose-dependent manner in the same model, and tumor cell apoptosis was increased by zoledronic acid in <em>bone</em> metastases in the 4T1 murine model of mammary carcinoma metastasis. Zoledronic acid at a dose of 1.0 microg/d for <em>10</em> days also reduced <em>bone</em> lesion area in a nude mouse model with existing <em>bone</em> metastases. Although bisphosphonates, including zoledronic acid, are able to induce apoptosis in tumor cells in vitro, studies in animal models to date have generally not shown a reduction in nonosseous tumor. Therefore, bisphosphonate-associated tumor reduction in <em>bone</em> is most likely mediated by osteoclast inhibition or is related to high local concentrations of bisphosphonates in the <em>bone</em> compartment.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP7) controls ureteric bud and collecting duct morphogenesis in a dose-dependent manner (Piscione TD, Yager TD, Gupta IR, Grinfeld B, Pei Y, Attisono L, Wrana JL, and Rosenblum ND. Am J Physiol Renal Physiol 273: F961-F975, 1997). We defined cellular and molecular mechanisms underlying these effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting tubule morphogenesis. Low-dose (0.25 nM) BMP7 significantly increased tubule number and cell proliferation. Similar to BMP2, high-dose (<em>10</em> nM) BMP7 inhibited cell proliferation and stimulated apoptosis. To define molecular mechanisms, we identified signaling events downstream of BMP7. High-dose BMP7, but not low-dose BMP7, activated Smad1 in mIMCD-3 cells. Moreover, the inhibitory effects of high-dose BMP7 and BMP2, but not the stimulatory effects of low-dose BMP7, on tubulogenesis and cell proliferation were significantly reduced in mIMCD-3 cells stably expressing Smad1(Delta458), a dominant negative mutant form of Smad1, but not in cells stably expressing wild-type Smad1. We conclude that BMP7 exerts dose-dependent effects on ureteric bud or collecting duct cell proliferation and apoptosis by signaling via Smad1-dependent and Smad1-independent pathways.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) signaling in diseases is the subject of an overwhelming array of studies. BMPs are excellent targets for treatment of various clinical disorders. Several BMPs have already been shown to be clinically beneficial in the treatment of a variety of conditions, including BMP-2 and BMP-7 that have been approved for clinical application in nonunion <em>bone</em> fractures and spinal fusions. With the use of BMPs increasingly accepted in spinal fusion surgeries, other therapeutic approaches targeting BMP signaling are emerging beyond applications to skeletal disorders. These approaches can further utilize next-generation therapeutic tools such as engineered BMPs and ex vivo- conditioned cell therapies. In this review, we focused to provide insights into such clinical potentials of BMPs in metabolic and vascular diseases, and in cancer. [BMB reports 2011; 44(<em>10</em>): 619-634].