BMP-2 Treatment Research Articles

Osteoblast derived extracellular vesicles induced by dexamethasone: A novel biomimetic tool for enhancing osteogenesis in vitro.

Extracellular vesicles (EVs) are newly appreciated communicators involved in intercellular crosstalk, and have emerged as a promising biomimetic tool for bone tissue regeneration, overcoming many of the limitations associated with cell-based therapies. However, the significance of osteoblast-derived extracellular vesicles on osteogenesis has not been fully established. In this present study, we aim to investigate the therapeutic potential of extracellular vesicles secreted from consecutive 14days of dexamethasone-stimulated osteoblasts (OB-EVDex) to act as a biomimetic tool for regulating osteogenesis, and to elucidate the underlying mechanisms. OB-EVdex treated groups are compared to the clinically used osteo-inductor of BMP-2 as control. Our findings revealed that OB-EVDex have a typical bilayer membrane nanostructure of, with an average diameter of 178 ± 21nm, and that fluorescently labeled OB-EVDex were engulfed by osteoblasts in a time-dependent manner. The proliferation, attachment, and viability capacities of OB-EVDex-treated osteoblasts were significantly improved when compared to untreated cells, with the highest proliferative rate observed in the OB-EVDex + BMP-2 group. Notably, combinations of OB-EVDex and BMP-2 markedly promoted osteogenic differentiation by positively upregulating osteogenesis-related gene expression levels of RUNX2, BGLAP, SPP1, SPARC, Col 1A1, and ALPL relative to BMP-2 or OB-EVDex treatment alone. Mineralization assays also showed greater pro-osteogenic potency after combined applications of OB-EVDex and BMP-2, as evidenced by a notable increase in mineralized nodules (calcium deposition) revealed by Alkaline Phosphatase (ALP), Alizarin Red Alizarin Red staining (ARS), and von Kossa staining. Therefore, our findings shed light on the potential of OB-EVDex as a new therapeutic option for enhancing osteogenesis.

The isochromosome 20q abnormality of pluripotent cells interrupts germ layer differentiation.

Chromosome 20 abnormalities are some of the most frequent genomic changes acquired by human pluripotent stem cell (hPSC) cultures worldwide. Yet their effects on differentiation remain largely unexplored. We investigated a recurrent abnormality also found on amniocentesis, the isochromosome 20q (iso20q), during a clinical retinal pigment epithelium differentiation. Here we show that the iso20q abnormality interrupts spontaneous embryonic lineage specification. Isogenic lines revealed that under conditions that promote the spontaneous differentiation of wild-type hPSCs, the iso20q variants fail to differentiate into primitive germ layers and to downregulate pluripotency networks, resulting in apoptosis. Instead, iso20q cells are highly biased for extra-embryonic/amnion differentiation following inhibition of DNMT3B methylation or BMP2 treatment. Finally, directed differentiation protocols can overcome the iso20q block. Our findings reveal in iso20q a chromosomal abnormality that impairs the developmental competency of hPSCs toward germ layers but not amnion, which models embryonic developmental bottlenecks in the presence of aberrations.

The method of sinus node-like pacemaker cells from human induced pluripotent stem cells by BMP and Wnt signaling.

The embryonic development of sinus nodes (SAN) is co-regulated by multiple signaling pathways. Among these, the bone morphogenetic protein (BMP) and Wnt signaling pathways are involved in the development of SAN. In this study, the effects of BMP and Wnt signaling on the differentiation of SAN-like pacemaker cells (SANLPCs) were investigated. Human induced pluripotent stem cells (hiPSCs) were divided into four groups: control, BMP4, CHIR-3, and BMP4 + CHIR (CHIR: a Wnt signaling activator). The samples were tested at day (D) 15 of differentiation. The final protocol for the activation of BMP signaling at D0-D3 and reactivation of Wnt signaling at D5-D7 in the differentiation of hiPSCs were determined. The results showed that the mRNA levels of pacemaker markers (TBX18, SHOX2, TBX3, HCN4, and HCN1) were higher in the BMP4 + CHIR group than in the control group, and working myocardial genes were downregulated. The immunofluorescence assay revealed that the expression of SHOX2 and HCN4 increased in the BMP4 + CHIR group compared to that in the other groups. In addition, the results of patch clamps revealed that a funny current of higher density and typical SAN action potentials were recorded, except in the control group, in which the L-type calcium current was higher in the BMP4 + CHIR group than in the other groups. Finally, the proportion of SANLPCs (cTnT+ NKX2.5-) was further enhanced by the combination of BMP4 and CHIR treatment. In summary, the combination of BMP and Wnt signaling promotes the differentiation of SANLPCs from hiPSCs.

Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

BMP-6 promotes type 2 immune response during enhancement of rat mandibular bone defect healing.

Bone morphogenetic proteins (BMPs) are used as key therapeutic agents for the treatment of difficult fractures. While their effects on osteoprogenitors are known, little is known about their effects on the immune system. We used permutations of BMP-6 (B), vascular endothelial growth factor (V), and Hedgehog signaling pathway activator smoothened agonist (S), to treat a rat mandibular defect and investigated healing outcomes at week 8, in correlation with the cellular landscape of the immune cells in the fracture callus at week 2. Maximum recruitment of immune cells to the fracture callus is known to occur at week 2. While the control, S, V, and VS groups remained as nonunions at week 8; all BMP-6 containing groups - B, BV, BS and BVS, showed near-complete to complete healing. This healing pattern was strongly associated with significantly higher ratios of CD4 T (CD45+CD3+CD4+) to putative CD8 T cells (CD45+CD3+CD4-), in groups treated with any permutation of BMP-6. Although, the numbers of putative M1 macrophages (CD45+CD3-CD11b/c+CD38high) were significantly lower in BMP-6 containing groups in comparison with S and VS groups, percentages of putative - Th1 cells or M1 macrophages (CD45+CD4+IFN-γ+) and putative - NK, NKT or cytotoxic CD8T cells (CD45+CD4-IFN-γ+) were similar in control and all treatment groups. Further interrogation revealed that the BMP-6 treatment promoted type 2 immune response by significantly increasing the numbers of CD45+CD3-CD11b/c+CD38low putative M2 macrophages, putative - Th2 cells or M2 macrophages (CD45+CD4+IL-4+) cells and putative - mast cells, eosinophils or basophils (CD45+CD4-IL-4+ cells). CD45- non-haematopoietic fractions of cells which encompass all known osteoprogenitor stem cells populations, were similar in control and treatment groups. This study uncovers previously unidentified regulatory functions of BMP-6 and shows that BMP-6 enhances fracture healing by not only acting on osteoprogenitor stem cells but also by promoting type 2 immune response.

The role of Smad4/TGFB signaling in tumorigenesis of pancreatic ductal adenocarcinoma (PDAC).

748 Background: Pancreatic cancer ranks 4th among cancers as a cause of death in the United States and 85- 90% of pancreatic cancer are pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated 2 major types of precursors to PDAC including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN); the mechanisms by which IPMNs give rise to PDAC are incompletely defined. We hypothesize that SMAD4 signaling is specifically involved in IPMN pathway with PanINs and IPMN having partially overlapping signaling pathways and molecular markers. Methods: We performed immunohistochemistry staining on different precursor lesions of mouse pancreatic tissue (PanIN precursor: Ptf1aCre LSL-KrasG12D; IPMN precursor: Ptf1aCreERT LSL- KrasG12D Smad4fl/fl treated with tamoxifen) using markers of different IPMN subtypes and signaling pathways known to be involved in PDAC including, MUC1, MUC2, MUC5AC, SMAD4, ERK1/2, SOX9, and YAP. We treated IPMN cells lines derived from Ptf1aCreERTM LSL-KrasG12D GnasR201C mice with TGF-β, BMP-2, or vehicle control and quantified RNA expression of Smad7, Serpine1, Id2, and normalized to Pmm1 expression. We performed a western blot to detect the protein expression of phospho-SMAD1, phospho-SMAD2, SMAD1, and SMAD2. Results: Both PanIN and IPMN mouse models exhibited strong expression of MUC5AC and MUC1, reflecting the pancreatobiliary subtype. The tumor cells in both models expressed phosphorylated substrates of protein kinase A, phosphorylated ERK1/2, and SOX9. Regional differences were noted for SOX9 signaling with IPMN demonstrating a greater expression around the outer edges of the lesion, compared to the center. PanIN model demonstrated a more prominent nuclear YAP staining. Treatment of BMP2 and TGF-β in pancreatic IPMN cells from Ptf1aCreERTM LSL-KrasG12D GnasR201C cell line significantly increased RNA levels of Serpine1 (2.4 and 199.6, respectively; n = 4; p < 0.05) and Smad7 (2.9 and 6.3, respectively; n = 4; p < 0.05). BMP2 treatment significantly increased RNA levels of Id2 (1.9; n = 4; p < 0.05) while TGF-β significantly decreased Id2 level (0.3; n = 4; p < 0.05). Phosphorylated-SMAD2 was detected only with TGF-β treatment while phosphorylated-SMAD1 was detected with vehicle, TGF-β, and BMP2 treatment with the highest level with BMP2 treatment. Conclusions: Knocking out Smad4, concurrent with activating KrasG12D mutation, induces a specific IPMN progression model that shares similar molecular markers as the PanIN progression model, shown in Kras mutation alone. SMAD4 signaling remains intact in IPMN lesions that arise from activating the KrasG12D GnasR201C mutation, possibly suggesting that Smad4 mutation induces the IPMN-PDAC progression model that differs from Gas-cAMP-PAK-YAP1 cascade found in GNAS signaling.

Abstract TP209: Role Of Endoglin In The Regulation Of Organic Anion Transporting Polypeptide 1a4 At The Blood-brain Barrier: Relevance To The Treatment Of Ischemic Stroke

Objectives: We have shown that transforming growth factor-β (TGF-β)/activin-like kinase 1 (ALK1) signaling at the blood-brain barrier (BBB) can regulate organic anion transporting polypeptide 1a4 (Oatp1a4), a transporter involved in brain uptake of drugs relevant to stroke pharmacotherapy (i.e., 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins)). To date, the involvement of endoglin (ENG), a co-receptor that can modulate the TGF-β/ALK1 pathway, in regulation of Oatp1a4 expression/activity has not been elucidated. The objective of this study is to evaluate whether ENG plays a role in the control of BBB transport mechanisms for stroke therapeutics. Methods: Our studies were conducted using the immortalized mouse brain endothelial cell line bEnd.3. Endoglin knockdown mutants were generated through lentiviral transfection of ENG-shRNA and validated using confocal microscopy and western blot analysis. Cells were then treated with bone morphogenetic protein-9 (BMP-9; 1 nM), a known TGF-β/ALK1 agonist. Oatp1a4 protein expression and transport activity were measured using western blot analysis and in vitro uptake assays using the known Oatp substrate drug [ 3 H]atorvastatin. Additionally, ENG expression in bEnd.3 cells was assessed under normoxic and oxygen/glucose deprivation (OGD) conditions (i.e., 1% O 2 in glucose-free medium). Results: BMP-9 treatment in non-transfected bEnd.3 cells resulted in increased Oatp1a4 protein expression and increased cellular uptake of [ 3 H]atorvastatin. These enhancements in Oatp1a4 functional expression were attenuated in ENG-shRNA knockdown bEnd.3 cells treated with BMP-9. Confocal microscopy and western blot analysis confirmed decreased ENG protein expression following shRNA transfection. Under OGD conditions, expression of ENG in bEnd.3 cells was significantly increased. Conclusions: Our data provide the first evidence for involvement of ENG in regulation of Oatp1a4 by TGF-β/ALK1 signaling in brain microvessel endothelial cells. These observations have clear implications for CNS delivery of drugs relevant to treatment of stroke. Studies are ongoing in our laboratory to fully evaluate ENG regulation of BBB transport mechanisms in the setting of ischemic stroke.

Histological Examination of the Effects of Thrombopoietin Mimetic Peptide (TMP) and High-Fat Diet on Femur Fracture Healing

Background and Hypothesis: In the US, 11.3% of the population are diabetic. Impaired bone healing is a complication of diabetes that dramatically impacts quality of life. Thus, it is imperative to find effective, low-risk treatments for patients that can accelerate fracture healing. We propose treatment of femur fractures using a thrombopoietin (TPO) analogue, TMP, will expedite healing, reduce adverse side effects compared to FDA-approved BMP-2, and improve quality of life of diabetic fracture patients.
 Experimental Design or Project Methods: Tie2CreERT+ mice were bred with Mplfl/fl mice to generate mice in which the TPO receptor (Mpl) was deleted in cells of the endothelial lineage (Tie2 expressing cells) following tamoxifen induction (3 consecutive daily 10mg/kg doses). Tie2CreERT+ ; Mplfl/fl and Tie2CreERT+ ; Mpl+/+ mice served as experimental and control mice, respectively. Eight-week-old male mice of both genotypes were placed on a low-fat diet (LFD) or high-fat-diet (HFD) for 12 weeks. One week prior to surgery, mice were injected with tamoxifen to induce Cre-recombination. Mice were then subjected to femur fracture and treated with saline or TMP (33nmol/kg/day) for the first week post-surgery. Mice were euthanized at 1-, 2-, and 4-week post-surgery and injured femurs were isolated for histological evaluation of the fracture callus size and composition.
 Results: To date only Tie2CreERT+ ; Mplfl/fl specimens have been processed. As expected, untreated HFD mice exhibited impaired fracture healing compared to similarly untreated LFD mice. As would also be expected, no differences were observed in fracture healing histological parameters between saline and TMP treated Tie2 CreERT+ ; Mplfl/fl mice at similar time points post-surgery.
 Conclusion and Potential Impact: While ongoing, this study explores the efficacy of using thrombopoietic agents for fracture healing in type 2 diabetes. If promising, thrombopoietic agents could replace, BMP-2 treatment, and may improve the quality of life for individuals experiencing impaired fracture healing.

Oxidative metabolism is impaired by phosphate deficiency during fracture healing and is mechanistically related to BMP induced chondrocyte differentiation

Prior studies of acute phosphate restriction during the endochondral phase of fracture healing showed delayed chondrocyte differentiation was mechanistically linked to decreased bone morphogenetic protein signaling. In the present study, transcriptomic analysis of fracture callus gene expression in three strains of mice was used to identify differentially expressed (FDR = q ≤ 0.05) genes in response to phosphate (Pi) restriction. Ontology and pathway analysis of these genes showed that independent of genetic background, a Pi-deficient diet downregulated (p = 3.16 × 10−23) genes associated with mitochondrial oxidative phosphorylation pathways as well as multiple other pathways of intermediate metabolism. Temporal clustering was used to identify co-regulation of these specific pathways. This analysis showed that specific Ox/Phos, tricarboxylic acid cycle, pyruvate dehydrogenase. Arginine, proline metabolism genes, and prolyl 4-hydroxylase were all co-regulated in response to dietary Pi restriction. The murine C3H10T½ mesenchymal stem cell line was used to assess the functional relationships between BMP2-induced chondrogenic differentiation, oxidative metabolism and extracellular matrix formation. BMP2-induced chondrogenic differentiation of C3H10T½ was carried out in culture media in the absence or presence of ascorbic acid, the necessary co-factor for proly hydroxylation, and in media with normal and 25 % phosphate levels. BMP2 treatment led to decreased proliferation, increased protein accumulation and increased collagen and aggrecan gene expression. Across all conditions, BMP2 increased total oxidative activity and ATP synthesis. Under all conditions, the presence of ascorbate further increased total protein accumulation, proly-hydroxylation and aggrecan gene expression, oxidative capacity and ATP production. Lower phosphate levels only diminished aggrecan gene expression with no other effects of metabolic activity being observed. These data suggest that dietary phosphate restriction controls endochondral growth in vivo indirectly through BMP signaling, which upregulates oxidative activity that is linked to overall protein production and collagen hydroxylation.

PDGF inhibits BMP2-induced bone healing

Bone regeneration depends on a pool of bone/cartilage stem/progenitor cells and signaling mechanisms regulating their differentiation. Using in vitro approach, we have shown that PDGF signaling through PDGFRβ inhibits BMP2-induced osteogenesis, and significantly attenuates expression of BMP2 target genes. We evaluated outcomes of treatment with two anabolic agents, PDGF and BMP2 using different bone healing models. Targeted deletion of PDGFRβ in αSMA osteoprogenitors, led to increased callus bone mass, resulting in improved biomechanical properties of fractures. In critical size bone defects BMP2 treatment increased proportion of osteoprogenitors, while the combined treatment of PDGF BB with BMP2 decreased progenitor number at the injury site. BMP2 treatment induced significant bone formation and increased number of osteoblasts, while in contrast combined treatment with PDGF BB decreased osteoblast numbers. This is in vivo study showing that PDGF inhibits BMP2-induced osteogenesis, but inhibiting PDGF signaling early in healing process does not improve BMP2-induced bone healing.

Natural history of Echinococcus granulosus microcyst development in long term in vitro culture and molecular and morphological changes induced by insulin and BMP-4.

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus is a disease of worldwide public health and economic importance. The determinants and underlying cellular mechanisms of CE development and fate in intermediate hosts are largely unknown. Hormones and cytokines such as insulin and BMP-4 are the key players in the development, differentiation, and apoptosis. In this study, we evaluated the long term natural history of E. granulosus microcysts in an vitro setting and the molecular and morphological changes induced by the growth factors, insulin and BMP4 during the development of metacestode stage of E. granulosus. E. granulosus protoscoleces were cultivated and the parasite development was followed in the long term mono-phasic culture for 105 days and the morphometric, molecular and immunohistochemical changes were evaluated, including the microcysts number and size, microcysts development and deformation rates as well as the markers of calcification (Alizarin Red staining) and apoptosis (BAX, BCL2, Caspase-3, Caspase-8 and TNF-α expression) in the microcysts. Also the biological, histological and molecular consequences of insulin and BMP-4 treatment on the parasite development were evaluated. Insulin and BMP-4 treatment of microcysts resulted in significant increase in microcyst formation, increased size, reduced apoptosis and deformation of the microcysts. Alizarin red staining of the microcysts treated with the insulin and BMP-4 confirmed that calcium deposition is significantly lower than the untreated microcysts. Also Alizarin Red staining and Immunohistochemistry of the microcysts indicates that calcium accumulation in deformed microcysts is higher than the normal ones on day 105. The microcysts began to wrinkle and the germinal layer was partially detached from the laminated layer on day 84. Results of the present study suggest that the degenerative changes in hydatid cysts can be slowed down by insulin and BMP-4, indicating that cellular factors and host hormones could contribute to the longevity of hydatid cysts. Significant evidences are provided suggesting that the microcysts cultivated in vitro can undergo calcification and apoptotic processes similar to what have been observed in the natural hydatid infection in the intermediate hosts.

Evaluating the Effects of Sustainable Chemical and Organic Fertilizers with Water Saving Practice on Corn Production and Soil Characteristics

The rapidly growing world population, water shortage, and food security are promising problems for sustainable agriculture. Farmers adopt higher irrigation and fertilizer applications to increase crop production resulting in environmental pollution. This study aimed to identify the long-term effects of intelligent water and fertilizers used in corn yield and soil nutrient status. A series of field experiments were conducted for six years with treatments as: farmer accustomed to fertilization used as control (CON), fertilizer decrement (KF), fertilizer decrement + water-saving irrigation (BMP1); combined application of organic and inorganic fertilizer + water-saving irrigation (BMP2), and combined application of controlled-release fertilizer (BMP3). A significant improvement was observed in soil organic matter (14.9%), nitrate nitrogen (106.7%), total phosphorus (23.9%), available phosphorus (26.2%), straw yield (44.8%), and grain yield (54.7%) with BMP2 treatment as compared to CON. The study concludes that integrating chemical and organic fertilizers with water-saving irrigation (BMP2) is a good approach to increasing corn productivity, ensuring water safety and improving soil health. The limitations of the current study include the identification of fertilizer type and its optimum dose, irrigation water type, and geographical position.

Endothelin-1 alters BMP signaling to promote proliferation of pulmonary artery smooth muscle cells.

Pulmonary arterial hypertension (PAH) is characterized by abnormal outgrowth of pulmonary artery smooth muscle cells (PASMCs) of the media. Abundant expression of endothelin-1 (ET-1) and activated p38 mitogen-activated protein kinase (p38MAPK) has been observed in PAH patients. p38MAPK has been implicated in cell proliferation. An unspecified disturbance in bone morphogenetic protein (BMP) signaling may be involved in the development of PAH. Type I receptors (BMPR1A and BMPR1B) and type II receptor (BMPR2) transduce signals via two distinct pathways, i.e., canonical and non-canonical pathways, activating Smad1/5/8 and p38MAPK, respectively. BMPR1B expression was previously reported to be enhanced in the PASMCs of patients with idiopathic PAH. BMP15 binds specifically to BMPR1B. We assessed the effects of ET-1 on BMP receptor expression and cell proliferation. BMP2 increased BMPR1B expression in human PASMCs after pretreatment with ET-1 in vitro. Although BMP2 alone did not affect PASMC proliferation, BMP2 treatment after ET-1 pretreatment significantly accelerated PASMC proliferation. PH-797804, a selective p38MAPK inhibitor, abrogated this proliferation. Similarly, after ET-1 pretreatment, BMP15 significantly accelerated the proliferation of PASMCs, whereas stimulation with BMP15 alone did not. In conclusion, in PASMCs, ET-1 exposure under pathological conditions alters BMP signaling to activate p38MAPK, resulting in cell proliferation.

The effects of Bone Morphogenetic Protein 4 on adult neural stem cell proliferation, differentiation and survival in an in vitro model of ischemic stroke

The subventricular zone (SVZ) of the lateral ventricles represents a main region where neural stem cells (NSCs) of the mature central nervous system (CNS) reside. Bone Morphogenetic Proteins (BMPs) are the largest subclass of the transforming growth factor-β (TGF-β) superfamily of ligands. BMP4 is one such member and plays important roles in adult NSC differentiation. However, the exact effects of BMP4 on SVZ adult NSCs in CNS ischemia are still unknown. Using oxygen and glucose deprivation (OGD) as an in vitro model of ischemia, we examined the behavior of adult NSCs. We observed that anoxia resulted in reduced viability of adult NSCs, and that BMP4 treatment clearly rescued apoptotic cell death following anoxia. Furthermore, BMP4 treatment exhibited a strong inhibitory effect on cellular proliferation of the adult NSCs in normoxic conditions. Moreover, such inhibitory effects of BMP4 treatment were also found in OGD conditions, despite the enhanced cellular proliferation of the adult NSCs that was observed under such ischemic conditions. Increased neuronal and astroglial commitment of adult NSCs were found in the OGD conditions, whereas a reduction in differentiated neurons and an increase in differentiated astrocytes were observed following BMP4 treatment. The present data indicate that BMP4 modulates proliferation and differentiation of SVZ-derived adult NSCs and promotes cell survival in the in vitro model of ischemic stroke.

Local administration of low doses of exogenous BMP2 and leptin promotes ectopic bone regeneration in leptin-deficient mice.

Obesity and leptin deficiency are associated with compromised bone regeneration. This study aims to investigate the role of locally administrated low-dose BMP2+leptin on bone regeneration in leptin-deficient obese (ob/ob) mice. Wildtype (WT) and ob/ob mice were divided into 3 groups (4mice/group): BMP2 (5μg) group, BMP2+low-dose leptin (1μg) group, and BMP2+high-dose leptin (2.5μg) group. WT mice were used as control mice. An equal size absorbable collagen sponge was prepared by loading the BMP2 or/and leptin and implanted subcutaneously. After 19 days, samples were collected and analyzed by micro-CT and H&E staining. No significant difference in bone regeneration among the three groups in WT mice. Quantification of newly formed bone parameters from micro-CT and H&E staining showed that low-dose BMP2 treatment formed less new bone in ob/ob mice compared to WT. BMP2+low-dose leptin treatment substantially rescued the compromised bone regeneration in ob/ob mice up to the level in WT mice. However, the BMP2 and high dose of leptin failed to rescue the compromised bone regeneration in ob/ob mice. Our findings suggest that a combination of the low-dose BMP2 and leptin could be a strategy to promote osteogenesis in obese populations with leptin deficiency.

RUNX2 Regulates Osteoblast Differentiation via the BMP4 Signaling Pathway

RUNX2 is a master osteogenic transcription factor, and mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Studies have revealed that RUNX2 is not only a downstream target of the bone morphogenetic protein (BMP) pathway but can also regulate the expression of BMPs. However, the underlying mechanism of the regulation of BMPs by RUNX2 remains unknown. In this project, we diagnosed a CCD patient with a 7.86-Mb heterozygous deletion on chromosome 6 containing all exons of RUNX2 by multiplex ligation-dependent probe amplification (MLPA) and whole-genome sequencing (WGS). Bone marrow mesenchymal stem cells (BMSCs) were further extracted from patient alveolar bone fragments (CCD-BMSCs), an excellent natural model to explore the possible mechanism. The osteogenic differentiation ability of CCD-BMSCs was severely affected by RUNX2 heterozygous deletion. Also, BMP4 decreased most in BMP ligands, and CHRDL1, a BMP antagonist, was abnormally elevated in CCD-BMSCs. Furthermore, BMP4 treatment essentially rescued the osteogenic capacity of CCD-BMSCs, and RUNX2 overexpression reversed the abnormal expression of BMP4 and CHRDL1. Notably, we constructed CRISPR/Cas9 Runx2+/m MC3T3-E1 cells, which simulated a variant in CCD-BMSCs, to exclude the interference of other gene deletions and the heterogeneity of the genetic background of primary cells, and verified all findings from the CCD-BMSCs. Moreover, the luciferase reporter experiment showed that RUNX2 could inhibit the transcription of CHRDL1. Through immunofluorescence, the inhibitory effect of CHRDL1 on BMP4/Smad signaling was confirmed in MC3T3-E1 cells. These results revealed that RUNX2 regulated the BMP4 pathway by inhibiting CHRDL1 transcription. We collectively identified a novel RUNX2/CHRDL1/BMP4 axis to regulate osteogenic differentiation and noted that BMP4 might be a valuable therapeutic option for treating bone diseases.

BMP4 Exerts Anti-Neurogenic Effect via Inducing Id3 during Aging.

Bone morphogenetic protein (BMP) signaling has been shown to be intimately associated with adult neurogenesis in the subventricular zone (SVZ) and subgranular zone (SGZ). Adult neurogenesis declines in aging rodents and primates. However, the role of BMP signaling in the age-related neurogenesis decline remains elusive and the effect of BMP4 on adult SVZ neurogenesis remains controversial. Here, the expression of BMP4 and its canonical effector phosphorylated-Smad1/5/8 (p-Smad1/5/8) in the murine SVZ and SGZ were found to be increased markedly with age. We identified Id3 as a major target of BMP4 in neuronal stem cells (NSCs) of both neurogenic regions, which exhibited a similar increase during aging. Intracerebroventricular infusion of BMP4 activated Smad1/5/8 phosphorylation and upregulated Id3 expression, which further restrained NeuroD1, leading to attenuated neurogenesis in both neurogenic regions and defective differentiation in the SGZ. Conversely, noggin, a potent inhibitor of BMP4, demonstrated opposing effects. In support of this, BMP4 treatment or lentiviral overexpression of Id3 resulted in decreased NeuroD1 protein levels in NSCs of both neurogenic regions and significantly inhibited neurogenesis. Thus, our findings revealed that the increased BMP4 signaling with age inhibited adult neurogenesis in both SVZ and SGZ, which may be attributed at least in part, to the changes in the Id3-NeuroD1 axis.

BMP9 reduces age-related bone loss in mice by inhibiting osteoblast senescence through Smad1-Stat1-P21 axis

Age-related osteoporosis is characterized by the accumulation of senescent osteoblastic cells in bone microenvironment and significantly reduced osteogenic differentiation. Clearing of the senescent cells is helpful to improve bone formation in aged mice. Bone morphogenetic protein 9 (BMP9), a multifunctional protein produced and secreted by liver, was reported to improve osteoporosis caused by estrogen withdrawal. However, the mechanism of BMP9 has not been fully elucidated, and its effect on senile osteoporosis has not been reported. This study reveals that BMP9 significantly increases bone mass and improves bone biomechanical properties in aged mice. Furthermore, BMP9 reduces expression of senescent genes in bone microenvironment, accompanied by decreased senescence-associated secretory phenotypes (SASPs) such as Ccl5, Mmp9, Hmgb1, Nfkb1, and Vcam1. In vitro, Bmp9 treatment inhibits osteoblast senescence through activating Smad1, which suppresses the transcriptional activity of Stat1, thereby inhibits P21 expression and SASPs production. Furthermore, inhibiting the Smad1 signal in vivo can reverse the inhibitory effect of BMP9 on Stat1 and downstream senescent genes, which eliminates the protection of BMP9 on age-related osteoporosis. These findings highlight the critical role of BMP9 on reducing age-related bone loss by inhibiting osteoblast senescence through Smad1-Stat1-P21 axis.

Characterization of Human Cardiac Progenitor Cell Secretome

Heart Disease (HD) remains the greatest cause of death worldwide. Due to the heart’s poor capacity to regenerate after damage, there is a critical need for treatments that can restore cardiac cell integrity and function in HD patients. Preclinical and clinical evidence has highlighted the potential role of Cardiac Progenitor Cells (CPCs) in regulation of cardiac repair after injury, with an emerging role of CPC secreted proteins in this process. We have been studying CPCs isolated from human left ventricular myocardium characterized by their high proliferative capacity in vitro. These cells (human highly proliferative clones, hHiPC) are characterized by high expression of CD105/Endoglin and show varying potential for differentiating into endothelial cells in vitro. When injected into the mouse myocardium after surgically induced myocardial infarction (MI), individual hHiPC clones show variable effects on heart remodeling. For instance, when clone 11 was injected into the infarct zone post‐MI we saw a trend towards improvement in the fractional shortening (%) compared to clone 22 and saline injected controls. We hypothesized that hHiPCs associated with improvement in cardiac remodeling after MI will have a secretome characterized by higher expression of pro‐angiogenic and pro‐proliferative proteins, and these are regulated by the BMP/CD105 pathway. An unbiased and global proteomics approach (LC‐MS/MS) was used in our lab to identify proteins secreted from hHiPCs pre‐treated with the CD105 ligand BMP9 (5 ng/mL) compared to non‐treated controls. Our secretome data identified ~700 proteins in which 57% were characterized from UniProt as having a signal peptide sequence. Several pro‐angiogenic secreted proteins were identified in clone 11 including CCN2, CXCL6, and TGFB2. We found a >2.5‐fold significant increase in the secretion of Sclerostin (SOST) in BMP9 treated hHiPC compared to non‐treated controls. SOST is a known BMP‐target protein and WNT inhibitor. Previous evidence has highlighted the role of WNT‐signaling in heart remodeling after myocardial induced injury, but the role of Sclerostin in this process has yet to be elucidated. To further characterize the relationship between BMP9 and the secretion of SOST we performed a Real‐Time q‐PCR and ELISA assay of cell lysates and conditioned media between BMP9 treated and non‐treated controls and found a dramatic increase in the expression of SOST in BMP9 treated clone 11. We also found an increase (4.5‐fold) in CD105 mRNA expression after BMP9 treatment. Similarly, using LC‐MS/MS we found a significant increase in CD105 expression (2.5‐fold, p‐value = 7.39x10‐6) in the membrane fraction of clone 11 compared to clone 22 indicating its potential role in positive heart remodeling effects. Future studies will be conducted in vivo to determine the role SOST/WNT signaling has in heart remodeling after ischemic injury. Further, the increase in pro‐angiogenic factors after BMP9 treatment may suggest the potential relevance of BMP9 treatment in cardiac progenitor cell secretome mediated repair and we will test this hypothesis in the future.

Preclinical Evaluation of BMP-9-Treated Human Bone-like Substitutes for Alveolar Ridge Preservation following Tooth Extraction.

The success of dental implant treatment after tooth extraction is generally maximized by preserving the alveolar ridge using cell-free biomaterials. However, these treatments can be associated with inflammatory reactions, leading to additional bone volume loss hampering dental implant positioning. Our group developed a self-assembled bone-like substitute constituted of osteogenically induced human adipose-derived stromal/stem cells (hASCs). We hypothesized that a bone morphogenetic protein (BMP) supplementation could improve the in vitro osteogenic potential of the bone-like substitute, which would subsequently translate into enhanced alveolar bone healing after tooth extraction. ASCs displayed a better osteogenic response to BMP-9 than to BMP-2 in monolayer cell culture, as shown by higher transcript levels of the osteogenic markers RUNX2, osterix (OSX/SP7), and alkaline phosphatase after three and six days of treatment. Interestingly, BMP-9 treatment significantly increased OSX transcripts and alkaline phosphatase activity, as well as pro-angiogenic angiopoietin-1 gene expression, in engineered bone-like substitutes after 21 days of culture. Alveolar bone healing was investigated after molar extraction in nude rats. Microcomputed tomography and histological evaluations revealed similar, or even superior, global alveolar bone preservation when defects were filled with BMP-9-treated bone-like substitutes for ten weeks compared to a clinical-grade biomaterial, with adequate gingival re-epithelialization in the absence of resorption.